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  • Synthetic Biology and Assembly Cloning  (46)
  • Atmosphere-ocean interaction  (19)
  • Oxford University Press  (46)
  • American Meteorological Society  (19)
  • MDPI Publishing
  • Nature Publishing Group
  • 2010-2014  (65)
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  • Oxford University Press  (46)
  • American Meteorological Society  (19)
  • MDPI Publishing
  • Nature Publishing Group
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  • 1
    Publication Date: 2022-05-25
    Description: Author Posting. © American Meteorological Society, 2013. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Physical Oceanography 43 (2013): 1589–1610, doi:10.1175/JPO-D-12-0173.1.
    Description: This study investigates the exchange of momentum between the atmosphere and ocean using data collected from four oceanic field experiments. Direct covariance estimates of momentum fluxes were collected in all four experiments and wind profiles were collected during three of them. The objective of the investigation is to improve parameterizations of the surface roughness and drag coefficient used to estimate the surface stress from bulk formulas. Specifically, the Coupled Ocean–Atmosphere Response Experiment (COARE) 3.0 bulk flux algorithm is refined to create COARE 3.5. Oversea measurements of dimensionless shear are used to investigate the stability function under stable and convective conditions. The behavior of surface roughness is then investigated over a wider range of wind speeds (up to 25 m s−1) and wave conditions than have been available from previous oversea field studies. The wind speed dependence of the Charnock coefficient α in the COARE algorithm is modified to , where m = 0.017 m−1 s and b = −0.005. When combined with a parameterization for smooth flow, this formulation gives better agreement with the stress estimates from all of the field programs at all winds speeds with significant improvement for wind speeds over 13 m s−1. Wave age– and wave slope–dependent parameterizations of the surface roughness are also investigated, but the COARE 3.5 wind speed–dependent formulation matches the observations well without any wave information. The available data provide a simple reason for why wind speed–, wave age–, and wave slope–dependent formulations give similar results—the inverse wave age varies nearly linearly with wind speed in long-fetch conditions for wind speeds up to 25 m s−1.
    Description: This work was funded by the National Science Foundation Grant OCE04-24536 as part of the CLIVAR Mode Water Dynamics Experiment (CLIMODE) and the Office of Naval Research Grant N00014-05-1-0139 as part of the CBLAST-LOW program.
    Description: 2014-02-01
    Keywords: Wind shear ; Wind stress ; Atmosphere-ocean interaction ; Fluxes ; Momentum ; Algorithms
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  • 2
    Publication Date: 2022-05-25
    Description: Author Posting. © American Meteorological Society, 2012. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 25 (2012): 2622–2651, doi:10.1175/JCLI-D-11-00301.1.
    Description: This study presents an overview of the El Niño–Southern Oscillation (ENSO) phenomenon and Pacific decadal variability (PDV) simulated in a multicentury preindustrial control integration of the NCAR Community Climate System Model version 4 (CCSM4) at nominal 1° latitude–longitude resolution. Several aspects of ENSO are improved in CCSM4 compared to its predecessor CCSM3, including the lengthened period (3–6 yr), the larger range of amplitude and frequency of events, and the longer duration of La Niña compared to El Niño. However, the overall magnitude of ENSO in CCSM4 is overestimated by ~30%. The simulated ENSO exhibits characteristics consistent with the delayed/recharge oscillator paradigm, including correspondence between the lengthened period and increased latitudinal width of the anomalous equatorial zonal wind stress. Global seasonal atmospheric teleconnections with accompanying impacts on precipitation and temperature are generally well simulated, although the wintertime deepening of the Aleutian low erroneously persists into spring. The vertical structure of the upper-ocean temperature response to ENSO in the north and south Pacific displays a realistic seasonal evolution, with notable asymmetries between warm and cold events. The model shows evidence of atmospheric circulation precursors over the North Pacific associated with the “seasonal footprinting mechanism,” similar to observations. Simulated PDV exhibits a significant spectral peak around 15 yr, with generally realistic spatial pattern and magnitude. However, PDV linkages between the tropics and extratropics are weaker than observed.
    Description: M. Alexander, A. Capotondi, and J. Scott’s participation was supported by a grant from the NSF Climate and Large-scale Dynamics Program. Y.-O. Kwon gratefully acknowledges support from a WHOI Heyman fellowship and a grant from the NSF Climate and Largescale Dynamics Program. The CESM project is supported by the National Science Foundation and the Office of Science (BER) of the U.S. Department of Energy.
    Description: 2012-10-15
    Keywords: Atmosphere-ocean interaction ; El Nino ; ENSO ; La Nina ; Pacific decadal oscillation ; Climate models
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  • 3
    Publication Date: 2022-05-25
    Description: Author Posting. © American Meteorological Society, 2012. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 25 (2012): 3515–3531, doi:10.1175/JCLI-D-11-00028.1.
    Description: The study examined global variability of air–sea sensible heat flux (SHF) from 1980 to 2009 and the large-scale atmospheric and ocean circulations that gave rise to this variability. The contribution of high-latitude wintertime SHF was identified, and the relative importance of the effect of the sea–air temperature difference versus the effect of wind on decadal SHF variability was analyzed using an empirical orthogonal function (EOF) approach. The study showed that global SHF anomalies are strongly modulated by SHF at high latitudes (poleward of 45°) during winter seasons. Decadal variability of global wintertime SHF can be reasonably represented by the sum of two leading EOF modes, namely, the boreal wintertime SHF in the northern oceans and the austral wintertime SHF in the southern oceans. The study also showed that global wintertime SHF is modulated by the prominent modes of the large-scale atmospheric circulation at high latitudes. The increase of global SHF in the 1990s is attributable to the strengthening of the Southern Hemisphere annular mode index, while the decrease of global SHF after 2000 is due primarily to the downward trend of the Arctic Oscillation index. This study identified the important effects of wind direction and speed on SHF variability. Changes in winds modify the sea–air temperature gradient by advecting cold and dry air from continents and by imposing changes in wind-driven oceanic processes that affect sea surface temperature (SST). The pattern of air temperature anomalies dominates over the pattern of SST anomalies and dictates the pattern of decadal SHF variability.
    Description: The study is supported by the NOAA Office of Climate Observations (OCO) and the WHOI Arctic Climate Initiative. X. Song acknowledges the support from the China Scholarship Council, National Natural Science Foundation of China (NSFC) (40930844, 40976004, and 40921004) and the Ministry of Education’s 111 Project (B07036).
    Description: 2012-11-15
    Keywords: Atmosphere-ocean interaction
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  • 4
    Publication Date: 2022-05-25
    Description: Author Posting. © American Meteorological Society, 2012. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 25 (2012): 3549–3565, doi:10.1175/JCLI-D-11-00320.1.
    Description: The recently released NCEP Climate Forecast System Reanalysis (CFSR) is used to examine the response to ENSO in the northeast tropical Pacific Ocean (NETP) during 1979–2009. The normally cool Pacific sea surface temperatures (SSTs) associated with wind jets through the gaps in the Central American mountains at Tehuantepec, Papagayo, and Panama are substantially warmer (colder) than the surrounding ocean during El Niño (La Niña) events. Ocean dynamics generate the ENSO-related SST anomalies in the gap wind regions as the surface fluxes damp the SSTs anomalies, while the Ekman heat transport is generally in quadrature with the anomalies. The ENSO-driven warming is associated with large-scale deepening of the thermocline; with the cold thermocline water at greater depths during El Niño in the NETP, it is less likely to be vertically mixed to the surface, particularly in the gap wind regions where the thermocline is normally very close to the surface. The thermocline deepening is enhanced to the south of the Costa Rica Dome in the Papagayo region, which contributes to the local ENSO-driven SST anomalies. The NETP thermocline changes are due to coastal Kelvin waves that initiate westward-propagating Rossby waves, and possibly ocean eddies, rather than by local Ekman pumping. These findings were confirmed with regional ocean model experiments: only integrations that included interannually varying ocean boundary conditions were able to simulate the thermocline deepening and localized warming in the NETP during El Niño events; the simulation with variable surface fluxes, but boundary conditions that repeated the seasonal cycle, did not.
    Description: This research was supported by grants from the NOAA office of Global Programs and the NSF Climate and Global Dynamics Division.
    Description: 2012-11-15
    Keywords: North Pacific Ocean ; Atmosphere-ocean interaction ; ENSO ; Thermocline circulation ; Waves, oceanic ; Ocean models
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  • 5
    Publication Date: 2022-05-25
    Description: Author Posting. © American Meteorological Society, 2014. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 27 (2014): 2405–2416, doi:10.1175/JCLI-D-13-00359.1.
    Description: Several recent studies utilizing global climate models predict that the Pacific Equatorial Undercurrent (EUC) will strengthen over the twenty-first century. Here, historical changes in the tropical Pacific are investigated using the Simple Ocean Data Assimilation (SODA) reanalysis toward understanding the dynamics and mechanisms that may dictate such a change. Although SODA does not assimilate velocity observations, the seasonal-to-interannual variability of the EUC estimated by SODA corresponds well with moored observations over a ~20-yr common period. Long-term trends in SODA indicate that the EUC core velocity has increased by 16% century−1 and as much as 47% century−1 at fixed locations since the mid-1800s. Diagnosis of the zonal momentum budget in the equatorial Pacific reveals two distinct seasonal mechanisms that explain the EUC strengthening. The first is characterized by strengthening of the western Pacific trade winds and hence oceanic zonal pressure gradient during boreal spring. The second entails weakening of eastern Pacific trade winds during boreal summer, which weakens the surface current and reduces EUC deceleration through vertical friction. EUC strengthening has important ecological implications as upwelling affects the thermal and biogeochemical environment. Furthermore, given the potential large-scale influence of EUC strength and depth on the heat budget in the eastern Pacific, the seasonal strengthening of the EUC may help reconcile paradoxical observations of Walker circulation slowdown and zonal SST gradient strengthening. Such a process would represent a new dynamical “thermostat” on CO2-forced warming of the tropical Pacific Ocean, emphasizing the importance of ocean dynamics and seasonality in understanding climate change projections.
    Description: EJDis supported by NSFGrantsOCE-1031971 and OCE-1233282. KBK is supported by NSF Grant OCE-1233282.
    Description: 2014-09-15
    Keywords: Tropics ; Currents ; Ocean dynamics ; Atmosphere-ocean interaction ; Climate variability ; Reanalysis data
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  • 6
    Publication Date: 2022-05-25
    Description: Author Posting. © American Meteorological Society, 2013. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 26 (2013): 9839–9859, doi:10.1175/JCLI-D-12-00647.1.
    Description: Spatial and temporal covariability between the atmospheric transient eddy heat fluxes (i.e., υ′T′ and υ′q′) in the Northern Hemisphere winter (January–March) and the paths of the Gulf Stream (GS), Kuroshio Extension (KE), and Oyashio Extension (OE) are examined based on an atmospheric reanalyses and ocean observations for 1979–2009. For the climatological winter mean, the northward heat fluxes by the synoptic (2–8 days) transient eddies exhibit canonical storm tracks with their maxima collocated with the GS and KE/OE. The intraseasonal (8 days–3 months) counterpart, while having overall similar amplitude, shows a spatial pattern with more localized maxima near the major orography and blocking regions. Lateral heat flux divergence by transient eddies as the sum of the two frequency bands exhibits very close coupling with the exact locations of the ocean fronts. Linear regression is used to examine the lead–lag relationship between interannual changes in the northward heat fluxes by the transient eddies and the meridional changes in the paths of the GS, KE, and OE, respectively. One to three years prior to the northward shifts of each ocean front, the atmospheric storm tracks shift northward and intensify, which is consistent with wind-driven changes of the ocean. Following the northward shifts of the ocean fronts, the synoptic storm tracks weaken in all three cases. The zonally integrated northward heat transport by the synoptic transient eddies increases by ~5% of its maximum mean value prior to the northward shift of each ocean front and decreases to a similar amplitude afterward.
    Description: Support from the National Aeronautics and Space Administration (NASA) Physical Oceanography Program (NNX09AF35G to TJ and Y-OK) and the Department of Energy (DOE) Climate and Environmental Sciences Division (DE-SC0007052 to Y-OK) is gratefully acknowledged.
    Description: 2014-06-15
    Keywords: Atmosphere-ocean interaction ; Eddies ; Energy transport ; Storm tracks ; Heat budgets/fluxes
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  • 7
    Publication Date: 2022-05-25
    Description: Author Posting. © American Meteorological Society, 2010. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Physical Oceanography 40 (2010): 2768–2777, doi:10.1175/2010JPO4461.1.
    Description: Although sustained observations yield a description of the mean equatorial current system from the western Pacific to the eastern terminus of the Tropical Atmosphere Ocean (TAO) array, a comprehensive observational dataset suitable for describing the structure and pathways of the Equatorial Undercurrent (EUC) east of 95°W does not exist and therefore climate models are unconstrained in a region that plays a critical role in ocean–atmosphere coupling. Furthermore, ocean models suggest that the interaction between the EUC and the Galápagos Islands (92°W) has a striking effect on the basic state and coupled variability of the tropical Pacific. To this end, the authors interpret historical measurements beginning with those made in conjunction with the discovery of the Pacific EUC in the 1950s, analyze velocity measurements from an equatorial TAO mooring at 85°W, and analyze a new dataset from archived shipboard ADCP measurements. Together, the observations yield a possible composite description of the EUC structure and pathways in the eastern equatorial Pacific that may be useful for model validation and guiding future observation.
    Description: Karnauskas acknowledges the WHOI Penzance Endowed Fund in Support of Assistant Scientists.
    Keywords: Atmosphere-ocean interaction ; Currents ; In situ observations ; Model evaluation/performance ; Pacific Ocean ; Tropics
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  • 8
    Publication Date: 2022-05-25
    Description: Author Posting. © American Meteorological Society, 2013. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 26 (2013): 9774–9790, doi:10.1175/JCLI-D-12-00862.1.
    Description: The influence of the Atlantic meridional overturning circulation (AMOC) variability on the atmospheric circulation is investigated in a control simulation of the NCAR Community Climate System Model, version 3 (CCSM3), where the AMOC evolves from an oscillatory regime into a red noise regime. In the latter, an AMOC intensification is followed during winter by a positive North Atlantic Oscillation (NAO). The atmospheric response is robust and controlled by AMOC-driven SST anomalies, which shift the heat release to the atmosphere northward near the Gulf Stream/North Atlantic Current. This alters the low-level atmospheric baroclinicity and shifts the maximum eddy growth northward, affecting the storm track and favoring a positive NAO. The AMOC influence is detected in the relation between seasonal upper-ocean heat content or SST anomalies and winter sea level pressure. In the oscillatory regime, no direct AMOC influence is detected in winter. However, an upper-ocean heat content anomaly resembling the AMOC footprint precedes a negative NAO. This opposite NAO polarity seems due to the southward shift of the Gulf Stream during AMOC intensification, displacing the maximum baroclinicity southward near the jet exit. As the mode has somewhat different patterns when using SST, the wintertime impact of the AMOC lacks robustness in this regime. However, none of the signals compares well with the observed influence of North Atlantic SST anomalies on the NAO because SST is dominated in CCSM3 by the meridional shifts of the Gulf Stream/North Atlantic Current that covary with the AMOC. Hence, although there is some potential climate predictability in CCSM3, it is not realistic.
    Description: Support from the NOAA Climate Program Office (Grant Number NA10OAR4310202) and the European Community 7th Framework Programme (FP7 2007-2013) under Grant Agreements GA212643 (THOR) and n.308299 (NACLIM) is gratefully acknowledged.
    Description: 2014-06-15
    Keywords: Atmosphere-ocean interaction ; North Atlantic Oscillation ; Thermohaline circulation ; Decadal variability
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  • 9
    Publication Date: 2022-05-25
    Description: Author Posting. © American Meteorological Society, 2013. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 26 (2013): 8476–8494, doi:10.1175/JCLI-D-12-00860.1.
    Description: Characteristics of atmospheric blocking in the Southern Hemisphere (SH) are explored in atmospheric general circulation model (AGCM) simulations with the Community Atmosphere Model, version 3, with a particular focus on the Australia–New Zealand sector. Preferred locations of blocking in SH observations and the associated seasonal cycle are well represented in the AGCM simulations, but the observed magnitude of blocking is underestimated throughout the year, particularly in late winter and spring. This is related to overly zonal flow due to an enhanced meridional pressure gradient in the model, which results in a decreased amplitude of the longwave trough/ridge pattern. A range of AGCM sensitivity experiments explores the effect on SH blocking of tropical heating, midlatitude sea surface temperatures, and land–sea temperature gradients created over the Australian continent during austral winter. The combined effects of tropical heating and extratropical temperature gradients are further explored in a configuration that is favorable for blocking in the Australia–New Zealand sector with warm SST anomalies to the north of Australia, cold to the southwest of Australia, warm to the southeast, and cool Australian land temperatures. The blocking-favorable configuration indicates a significant strengthening of the subtropical jet and a reduction in midlatitude flow, which results from changes in the thermal wind. While these overall changes in mean climate, predominantly forced by the tropical heating, enhance blocking activity, the magnitude of atmospheric blocking compared to observations is still underestimated. The blocking-unfavorable configuration with surface forcing anomalies of opposite sign results in a weakening subtropical jet, enhanced midlatitude flow, and significantly reduced blocking.
    Description: C.C.U. received support from the Australian Research Council through funding awarded to the Centre of Excellence for Climate System Science and the Penzance Endowed Fund at WHOI. P.C.M., M.J.P., and J.S.R. were funded by the CSIRO Climate Adaptation Flagship and the Managing Climate Variability R&D Program.
    Description: 2014-05-01
    Keywords: Australia ; Southern Hemisphere ; Atmosphere-ocean interaction ; Atmospheric circulation ; Blocking ; General circulation models
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  • 10
    Publication Date: 2022-05-25
    Description: Author Posting. © American Meteorological Society, 2010. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 23 (2010): 3146–3154, doi:10.1175/2010JCLI3310.1.
    Description: The Atlantic meridional overturning circulation (AMOC) simulated in various ocean-only and coupled atmosphere–ocean numerical models often varies in time because of either forced or internal variability. The path of the Gulf Stream (GS) is one diagnostic variable that seems to be sensitive to the amplitude of the AMOC, yet previous modeling studies show a diametrically opposed relationship between the two variables. In this note this issue is revisited, bringing together ocean observations and comparisons with the GFDL Climate Model version 2.1 (CM2.1), both of which suggest a more southerly (northerly) GS path when the AMOC is relatively strong (weak). Also shown are some examples of possible diagnostics to compare various models and observations on the relationship between shifts in GS path and changes in AMOC strength in future studies.
    Description: We wish to acknowledge support (TJ) from WHOI’s Paul Fye Chair and NASA (NNXZX09AF35G) and to NOAA/OAR (RZ) for this work.
    Keywords: Sea surface temperature ; Meridional overturning circulation ; Gyres ; Coupled models ; Atmosphere-ocean interaction
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  • 11
    Publication Date: 2022-05-25
    Description: Author Posting. © American Meteorological Society, 2012. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 25 (2012): 6743–6755, doi:10.1175/JCLI-D-11-00549.1.
    Description: From 1969 to 1971 convection in the Labrador Sea shut down, thus interrupting the formation of the intermediate/dense water masses. The shutdown has been attributed to the surface freshening induced by the Great Salinity Anomaly (GSA), a freshwater anomaly in the subpolar North Atlantic. The abrupt resumption of convection in 1972, in contrast, is attributed to the extreme atmospheric forcing of that winter. Here oceanic and atmospheric data collected in the Labrador Sea at Ocean Weather Station Bravo and a one-dimensional mixed layer model are used to examine the causes of the shutdown and resumption of convection in detail. These results highlight the tight coupling of the ocean and atmosphere in convection regions and the need to resolve both components to correctly represent convective processes in the ocean. They are also relevant to present-day conditions given the increased ice melt in the Arctic Ocean and from the Greenland Ice Sheet. The analysis herein shows that the shutdown was initiated by the GSA-induced freshening as well as the mild 1968/69 winter. After the shutdown had begun, however, the continuing lateral freshwater flux as well as two positive feedbacks [both associated with the sea surface temperature (SST) decrease due to lack of convective mixing with warmer subsurface water] further inhibited convection. First, the SST decrease reduced the heat flux to the atmosphere by reducing the air–sea temperature gradient. Second, it further reduced the surface buoyancy loss by reducing the thermal expansion coefficient of the surface water. In 1972 convection resumed because of both the extreme atmospheric forcing and advection of saltier waters into the convection region.
    Description: This research was funded by a grant from the NWO/SRON User Support Programme Space Research. FS acknowledges support from OCE- 0850416 and NOAA NA08OAR4310569.
    Description: 2013-04-01
    Keywords: Atmosphere-ocean interaction ; Intermediate waters ; Oceanic variability
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  • 12
    Publication Date: 2022-05-26
    Description: Author Posting. © American Meteorological Society, 2014. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 27 (2014): 1343–1363, doi:10.1175/JCLI-D-13-00148.1.
    Description: The climate of West Antarctica is strongly influenced by remote forcing from the tropical Pacific. For example, recent surface warming over West Antarctica reflects atmospheric circulation changes over the Amundsen Sea, driven by an atmospheric Rossby wave response to tropical sea surface temperature (SST) anomalies. Here, it is demonstrated that tropical Pacific SST anomalies also influence the source and transport of marine-derived aerosols to the West Antarctic Ice Sheet. Using records from four firn cores collected along the Amundsen coast of West Antarctica, the relationship between sea ice–modulated chemical species and large-scale atmospheric variability in the tropical Pacific from 1979 to 2010 is investigated. Significant correlations are found between marine biogenic aerosols and sea salts, and SST and sea level pressure in the tropical Pacific. In particular, La Niña–like conditions generate an atmospheric Rossby wave response that influences atmospheric circulation over Pine Island Bay. Seasonal regression of atmospheric fields on methanesulfonic acid (MSA) reveals a reduction in onshore wind velocities in summer at Pine Island Bay, consistent with enhanced katabatic flow, polynya opening, and coastal dimethyl sulfide production. Seasonal regression of atmospheric fields on chloride (Cl−) reveals an intensification in onshore wind velocities in winter, consistent with sea salt transport from offshore source regions. Both the source and transport of marine aerosols to West Antarctica are found to be modulated by similar atmospheric dynamics in response to remote forcing. Finally, the regional ice-core array suggests that there is both a temporally and a spatially varying response to remote tropical forcing.
    Description: This research was supported by an award from the Department of Energy Office of Science Graduate Fellowship Program (DOE SCGF) to ASC, a James E. and Barbara V. Moltz Research Fellowship to SBD, and grants from NSF-OPP (ANT- 0632031 and ANT-0631973), NSF-MRI (EAR- 1126217), and the NASA Cryosphere Program (NNX10AP09G), and a WHOI Andrew W. Mellon Foundation Award for Innovative Research.
    Description: 2014-08-01
    Keywords: Antarctica ; Sea ice ; Teleconnections ; Atmosphere-ocean interaction ; Climate records ; Interannual variability
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  • 13
    Publication Date: 2022-05-26
    Description: Author Posting. © American Meteorological Society, 2013. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Atmospheric and Oceanic Technology 39 (2013): 450–469, doi:10.1175/JTECH-D-12-00078.1.
    Description: A surface mooring was deployed in the Gulf Stream for 15 months to investigate the role of air–sea interaction in mode water formation and other processes. The accuracies of the near-surface meteorological and oceanographic measurements are investigated. In addition, the impacts of these measurement errors on the estimation and study of the air–sea fluxes in the Gulf Stream are discussed. Pre- and postdeployment calibrations together with in situ comparison between shipboard and moored sensors supported the identification of biases due to sensor drifts, sensor electronics, and calibration errors. A postdeployment field study was used to further investigate the performance of the wind sensors. The use of redundant sensor sets not only supported the filling of data gaps but also allowed an examination of the contribution of random errors. Air–sea fluxes were also analyzed and computed from both Coupled Ocean–Atmosphere Response Experiment (COARE) bulk parameterization and using direct covariance measurements. The basic conclusion is that the surface buoy deployed in the Gulf Stream to support air–sea interaction research was successful, providing an improved 15-month record of surface meteorology, upper-ocean variability, and air–sea fluxes with known accuracies. At the same time, the coincident deployment of mean meteorological and turbulent flux sensors proved to be a successful strategy to certify the validity of the bulk formula fluxes over the midrange of wind speeds and to support further work to address the present shortcomings of the bulk formula methods at the low and high wind speeds.
    Description: The National Science Foundation (Grant OCE04-24536) funded this work, as part of the CLIVAR Mode Water Dynamics Experiment (CLIMODE). The Vetlesen Foundation is also acknowledged for the early support of S. Bigorre.
    Description: 2013-09-01
    Keywords: Atmosphere-ocean interaction ; Buoy observations
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  • 14
    Publication Date: 2022-05-26
    Description: Author Posting. © American Meteorological Society, 2014. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 27 (2014): 2842–2860, doi:10.1175/JCLI-D-13-00227.1.
    Description: Mooring measurements from the Kuroshio Extension System Study (June 2004–June 2006) and from the ongoing Kuroshio Extension Observatory (June 2004–present) are combined with float measurements of the Argo network to study the variability of the North Pacific Subtropical Mode Water (STMW) across the entire gyre, on time scales from days, to seasons, to a decade. The top of the STMW follows a seasonal cycle, although observations reveal that it primarily varies in discrete steps associated with episodic wind events. The variations of the STMW bottom depth are tightly related to the sea surface height (SSH), reflecting mesoscale eddies and large-scale variations of the Kuroshio Extension and recirculation gyre systems. Using the observed relationship between SSH and STMW, gridded SSH products and in situ estimates from floats are used to construct weekly maps of STMW thickness, providing nonbiased estimates of STMW total volume, annual formation and erosion volumes, and seasonal and interannual variability for the past decade. Year-to-year variations are detected, particularly a significant decrease of STMW volume in 2007–10 primarily attributable to a smaller volume formed. Variability of the heat content in the mode water region is dominated by the seasonal cycle and mesoscale eddies; there is only a weak link to STMW on interannual time scales, and no long-term trends in heat content and STMW thickness between 2002 and 2011 are detected. Weak lagged correlations among air–sea fluxes, oceanic heat content, and STMW thickness are found when averaged over the northwestern Pacific recirculation gyre region.
    Description: This work was sponsored by the National Science Foundation (Grants OCE-0220161, OCE-0825152, and OCE-0827125).
    Description: 2014-10-15
    Keywords: Atmosphere-ocean interaction ; Mesoscale processes ; Mesoscale systems ; Ocean dynamics ; Eddies ; Water masses
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  • 15
    Publication Date: 2022-05-26
    Description: Author Posting. © American Meteorological Society, 2014. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 27 (2014): 3298–3317, doi:JCLI-D-12-00700.1.
    Description: The subpolar North Atlantic is a center of variability of ocean properties, wind stress curl, and air–sea exchanges. Observations and hindcast simulations suggest that from the early 1970s to the mid-1990s the subpolar gyre became fresher while the gyre and meridional circulations intensified. This is opposite to the relationship of freshening causing a weakened circulation, most often reproduced by climate models. The authors hypothesize that both these configurations exist but dominate on different time scales: a fresher subpolar gyre when the circulation is more intense, at interannual frequencies (configuration A), and a saltier subpolar gyre when the circulation is more intense, at longer periods (configuration B). Rather than going into the detail of the mechanisms sustaining each configuration, the authors’ objective is to identify which configuration dominates and to test whether this depends on frequency, in preindustrial control runs of five climate models from phase 5 of the Coupled Model Intercomparison Project (CMIP5). To this end, the authors have developed a novel intercomparison method that enables analysis of freshwater budget and circulation changes in a physical perspective that overcomes model specificities. Lag correlations and a cross-spectral analysis between freshwater content changes and circulation indices validate the authors’ hypothesis, as configuration A is only visible at interannual frequencies while configuration B is mostly visible at decadal and longer periods, suggesting that the driving role of salinity on the circulation depends on frequency. Overall, this analysis underscores the large differences among state-of-the-art climate models in their representations of the North Atlantic freshwater budget.
    Description: JD and RC were funded by NSF through Project 0751896. JD was also funded by IFREMER through project RICCO.
    Description: 2014-11-01
    Keywords: Atmosphere-ocean interaction ; Freshwater ; Climate models ; Model comparison ; Climate variability ; North Atlantic Oscillation
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  • 16
    Publication Date: 2022-05-26
    Description: Author Posting. © American Meteorological Society, 2014. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Physical Oceanography 44 (2014): 2569–2587, doi:10.1175/JPO-D-14-0026.1.
    Description: This paper describes the occurrence of diurnal restratification events found in the southeast trade wind regime off northern Chile. This is a region where persistent marine stratus clouds are found and where there is a less than complete understanding of the dynamics that govern the maintenance of the sea surface temperature. A surface mooring deployed in the region provides surface meteorological, air–sea flux, and upper-ocean temperature, salinity, and velocity data. In the presence of steady southeast trade winds and strong evaporation, a warm, salty surface mixed layer is found in the upper ocean. During the year, these trade winds, at times, drop dramatically and surface heating leads to the formation of shallow, warm diurnal mixed layers over one to several days. At the end of such a low wind period, mean sea surface temperature is warmer. Though magnitudes of the individual diurnal warming events are consistent with local forcing, as judged by running a one-dimensional model, the net warming at the end of a low wind event is more difficult to predict. This is found to stem from differences between the observed and predicted near-inertial shear and the depths over which the warmed water is distributed. As a result, the evolution of SST has a dependency on these diurnal restratification events and on near-surface processes that govern the depth over which the heat gained during such events is distributed.
    Description: RAW was supported by the NOAA Climate Program Office. SM and AT were supported by NASA Grant NNX12AD47G,ONR Grant N000140910196, and NSF-OCE 0928138 RAW.
    Description: 2015-03-01
    Keywords: Atm/Ocean Structure/ Phenomena ; Atmosphere-ocean interaction ; Boundary layer ; Diurnal effects ; Mixed layer
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  • 17
    Publication Date: 2022-05-26
    Description: Author Posting. © American Meteorological Society, 2014. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 27 (2014): 3596–3618, doi:10.1175/JCLI-D-13-00070.1.
    Description: Estimates of the recent mean and time varying water mass transformation rates associated with North Atlantic surface-forced overturning are presented. The estimates are derived from heat and freshwater surface fluxes and sea surface temperature fields from six atmospheric reanalyses—the Japanese 25-yr Reanalysis (JRA), the NCEP–NCAR reanalysis (NCEP1), the NCEP–U.S. Department of Energy (DOE) reanalysis (NCEP2), the European Centre for Medium-Range Weather Forecasts (ECMWF) Interim Re-Analysis (ERA-I), the Climate Forecast System Reanalysis (CFSR), and the Modern-Era Reanalysis for Research and Applications (MERRA)—together with sea surface salinity fields from two globally gridded datasets (World Ocean Atlas and Met Office EN3 datasets). The resulting 12 estimates of the 1979–2007 mean surface-forced streamfunction all depict a subpolar cell, with maxima north of 45°N, near σ = 27.5 kg m−3, and a subtropical cell between 20° and 40°N, near σ = 26.1 kg m−3. The mean magnitude of the subpolar cell varies between 12 and 18 Sv (1 Sv ≡ 106 m3 s−1), consistent with estimates of the overturning circulation from subsurface observations. Analysis of the thermal and haline components of the surface density fluxes indicates that large differences in the inferred low-latitude circulation are largely a result of the biases in reanalysis net heat flux fields, which range in the global mean from −13 to 19 W m−2. The different estimates of temporal variability in the subpolar cell are well correlated with each other. This suggests that the uncertainty associated with the choice of reanalysis product does not critically limit the ability of the method to infer the variability in the subpolar overturning. In contrast, the different estimates of subtropical variability are poorly correlated with each other, and only a subset of them captures a significant fraction of the variability in independently estimated North Atlantic Subtropical Mode Water volume.
    Description: JPG is funded by UK Natural Environment Research Council New Investigator Grant NE/I001654/1. Y-OK was supported by the U.S. National Science Foundation under Grant OCE-0424492. RJB is supported by a fellowship from the UK National Centre for Earth Observation.
    Description: 2014-11-15
    Keywords: Atmosphere-ocean interaction ; Meridional overturning circulation ; Ocean circulation
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  • 18
    Publication Date: 2022-05-26
    Description: Author Posting. © American Meteorological Society, 2014. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 27 (2014): 2861–2885, doi:10.1175/JCLI-D-13-00437.1.
    Description: The representation of the El Niño–Southern Oscillation (ENSO) under historical forcing and future projections is analyzed in 34 models from the Coupled Model Intercomparison Project phase 5 (CMIP5). Most models realistically simulate the observed intensity and location of maximum sea surface temperature (SST) anomalies during ENSO events. However, there exist systematic biases in the westward extent of ENSO-related SST anomalies, driven by unrealistic westward displacement and enhancement of the equatorial wind stress in the western Pacific. Almost all CMIP5 models capture the observed asymmetry in magnitude between the warm and cold events (i.e., El Niños are stronger than La Niñas) and between the two types of El Niños: that is, cold tongue (CT) El Niños are stronger than warm pool (WP) El Niños. However, most models fail to reproduce the asymmetry between the two types of La Niñas, with CT stronger than WP events, which is opposite to observations. Most models capture the observed peak in ENSO amplitude around December; however, the seasonal evolution of ENSO has a large range of behavior across the models. The CMIP5 models generally reproduce the duration of CT El Niños but have biases in the evolution of the other types of events. The evolution of WP El Niños suggests that the decay of this event occurs through heat content discharge in the models rather than the advection of SST via anomalous zonal currents, as seems to occur in observations. No consistent changes are seen across the models in the location and magnitude of maximum SST anomalies, frequency, or temporal evolution of these events in a warmer world.
    Description: 2014-10-15
    Keywords: Atmosphere-ocean interaction ; Climate change ; Climate variability ; ENSO ; Climate models ; Model evaluation/performance
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  • 19
    Publication Date: 2022-05-26
    Description: Author Posting. © American Meteorological Society, 2012. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 25 (2012): 7781–7801, doi:10.1175/JCLI-D-11-00442.1.
    Description: Air–sea fluxes from the Community Climate System Model version 4 (CCSM4) are compared with the Coordinated Ocean-Ice Reference Experiment (CORE) dataset to assess present-day mean biases, variability errors, and late twentieth-century trend differences. CCSM4 is improved over the previous version, CCSM3, in both air–sea heat and freshwater fluxes in some regions; however, a large increase in net shortwave radiation into the ocean may contribute to an enhanced hydrological cycle. The authors provide a new baseline for assessment of flux variance at annual and interannual frequency bands in future model versions and contribute a new metric for assessing the coupling between the atmospheric and oceanic planetary boundary layer (PBL) schemes of any climate model. Maps of the ratio of CCSM4 variance to CORE reveal that variance on annual time scales has larger error than on interannual time scales and that different processes cause errors in mean, annual, and interannual frequency bands. Air temperature and specific humidity in the CCSM4 atmospheric boundary layer (ABL) follow the sea surface conditions much more closely than is found in CORE. Sensible and latent heat fluxes are less of a negative feedback to sea surface temperature warming in the CCSM4 than in the CORE data with the model’s PBL allowing for more heating of the ocean’s surface.
    Description: The CESM project is supported by the National Science Foundation and the Office of Science (BER) of the U.S. Department of Energy. S. Stevensonwas supported byNASAGrantNNX09A020H and B. Fox-Kemper by Grants NSF 0934737 and NASA NNX09AF38G.
    Description: 2013-05-15
    Keywords: Atmosphere-ocean interaction ; Boundary layer ; Sea surface temperature ; Climate models ; Coupled models ; Model evaluation/performance
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  • 20
    Publication Date: 2013-09-26
    Description: In reverse genetics, a gene’s function is elucidated through targeted modifications in the coding region or associated DNA cis -regulatory elements. To this purpose, recently developed customizable transcription activator-like effector nucleases (TALENs) have proven an invaluable tool, allowing introduction of double-strand breaks at predetermined sites in the genome. Here we describe a practical and efficient method for the targeted genome engineering in Drosophila . We demonstrate TALEN-mediated targeted gene integration and efficient identification of mutant flies using a traceable marker phenotype. Furthermore, we developed an easy TALEN assembly (easyT) method relying on simultaneous reactions of DNA Bae I digestion and ligation, enabling construction of complete TALENs from a monomer unit library in a single day. Taken together, our strategy with easyT and TALEN-plasmid microinjection simplifies mutant generation and enables isolation of desired mutant fly lines in the F 1 generation.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 21
    Publication Date: 2013-06-08
    Description: Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ~40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 22
    Publication Date: 2013-06-08
    Description: We describe a new cell-free protein synthesis (CFPS) method for site-specific incorporation of non-natural amino acids (nnAAs) into proteins in which the orthogonal tRNA (o-tRNA) and the modified protein (i.e. the protein containing the nnAA) are produced simultaneously. Using this method, 0.9–1.7 mg/ml of modified soluble super-folder green fluorescent protein (sfGFP) containing either p -azido- l -phenylalanine (pAzF) or p -propargyloxy- l -phenylalanine (pPaF) accumulated in the CFPS solutions; these yields correspond to 50–88% suppression efficiency. The o-tRNA can be transcribed either from a linearized plasmid or from a crude PCR product. Comparison of two different o-tRNAs suggests that the new platform is not limited by Ef-Tu recognition of the acylated o-tRNA at sufficiently high o-tRNA template concentrations. Analysis of nnAA incorporation across 12 different sites in sfGFP suggests that modified protein yields and suppression efficiencies (i.e. the position effect) do not correlate with any of the reported trends. Sites that were ineffectively suppressed with the original o-tRNA were better suppressed with an optimized o-tRNA (o-tRNA opt ) that was evolved to be better recognized by Ef-Tu. This new platform can also be used to screen scissile ribozymes for improved catalysis.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 23
    Publication Date: 2012-09-27
    Description: Genome-scale engineering of living organisms requires precise and economical methods to efficiently modify many loci within chromosomes. One such example is the directed integration of chemically synthesized single-stranded deoxyribonucleic acid (oligonucleotides) into the chromosome of Escherichia coli during replication. Herein, we present a general co-selection strategy in multiplex genome engineering that yields highly modified cells. We demonstrate that disparate sites throughout the genome can be easily modified simultaneously by leveraging selectable markers within 500 kb of the target sites. We apply this technique to the modification of 80 sites in the E. coli genome.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 24
    Publication Date: 2012-06-06
    Description: A chemistry-based artificial restriction DNA cutter (ARCUT) was recently prepared from Ce(IV)/EDTA complex and a pair of pseudo-complementary peptide nucleic acids. This cutter has freely tunable scission-site and site specificity. In this article, homologous recombination (HR) in human cells was promoted by cutting a substrate DNA with ARCUT, and the efficiency of this bioprocess was optimized by various chemical and biological approaches. Of two kinds of terminal structure formed by ARCUT, 3'-overhang termini provided by 1.7-fold higher efficiency than 5'-overhang termini. A longer homology length (e.g. 698 bp) was about 2-fold more favorable than shorter one (e.g. 100 bp). When the cell cycle was synchronized to G2/M phase with nocodazole, the HR was promoted by about 2-fold. Repression of the NHEJ-relevant proteins Ku70 and Ku80 by siRNA increased the efficiency by 2- to 3-fold. It was indicated that appropriate combination of all these chemical and biological approaches should be very effective to promote ARCUT-mediated HR in human cells.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 25
    Publication Date: 2012-04-24
    Description: We describe a novel cloning method termed SLiCE (Seamless L i gation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 26
    Publication Date: 2012-04-24
    Description: Targeted gene addition to mammalian genomes is central to biotechnology, basic research and gene therapy. For example, gene targeting to the ROSA26 locus by homologous recombination in embryonic stem cells is commonly used for mouse transgenesis to achieve ubiquitous and persistent transgene expression. However, conventional methods are not readily adaptable to gene targeting in other cell types. The emerging zinc finger nuclease (ZFN) technology facilitates gene targeting in diverse species and cell types, but an optimal strategy for engineering highly active ZFNs is still unclear. We used a modular assembly approach to build ZFNs that target the ROSA26 locus. ZFN activity was dependent on the number of modules in each zinc finger array. The ZFNs were active in a variety of cell types in a time- and dose-dependent manner. The ZFNs directed gene addition to the ROSA26 locus, which enhanced the level of sustained gene expression, the uniformity of gene expression within clonal cell populations and the reproducibility of gene expression between clones. These ZFNs are a promising resource for cell engineering, mouse transgenesis and pre-clinical gene therapy studies. Furthermore, this characterization of the modular assembly method provides general insights into the implementation of the ZFN technology.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 27
    Publication Date: 2012-05-13
    Description: A simple approach for creating libraries of circularly permuted proteins is described that is called PERMutation Using Transposase Engineering (PERMUTE). In PERMUTE, the transposase MuA is used to randomly insert a minitransposon that can function as a protein expression vector into a plasmid that contains the open reading frame (ORF) being permuted. A library of vectors that express different permuted variants of the ORF-encoded protein is created by: (i) using bacteria to select for target vectors that acquire an integrated minitransposon; (ii) excising the ensemble of ORFs that contain an integrated minitransposon from the selected vectors; and (iii) circularizing the ensemble of ORFs containing integrated minitransposons using intramolecular ligation. Construction of a Thermotoga neapolitana adenylate kinase (AK) library using PERMUTE revealed that this approach produces vectors that express circularly permuted proteins with distinct sequence diversity from existing methods. In addition, selection of this library for variants that complement the growth of Escherichia coli with a temperature-sensitive AK identified functional proteins with novel architectures, suggesting that PERMUTE will be useful for the directed evolution of proteins with new functions.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 28
    Publication Date: 2012-02-28
    Description: Synthetic scaffolds that permit spatial and temporal organization of enzymes in living cells are a promising post-translational strategy for controlling the flow of information in both metabolic and signaling pathways. Here, we describe the use of plasmid DNA as a stable, robust and configurable scaffold for arranging biosynthetic enzymes in the cytoplasm of Escherichia coli . This involved conversion of individual enzymes into custom DNA-binding proteins by genetic fusion to zinc-finger domains that specifically bind unique DNA sequences. When expressed in cells that carried a rationally designed DNA scaffold comprising corresponding zinc finger binding sites, the titers of diverse metabolic products, including resveratrol, 1,2-propanediol and mevalonate were increased as a function of the scaffold architecture. These results highlight the utility of DNA scaffolds for assembling biosynthetic enzymes into functional metabolic structures. Beyond metabolism, we anticipate that DNA scaffolds may be useful in sequestering different types of enzymes for specifying the output of biological signaling pathways or for coordinating other assembly-line processes such as protein folding, degradation and post-translational modifications.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 29
    Publication Date: 2014-03-13
    Description: To reveal the full potential of human pluripotent stem cells, new methods for rapid, site-specific genomic engineering are needed. Here, we describe a system for precise genetic modification of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We identified a novel human locus, H11 , located in a safe, intergenic, transcriptionally active region of chromosome 22, as the recipient site, to provide robust, ubiquitous expression of inserted genes. Recipient cell lines were established by site-specific placement of a ‘landing pad’ cassette carrying attP sites for phiC31 and Bxb1 integrases at the H11 locus by spontaneous or TALEN-assisted homologous recombination. Dual integrase cassette exchange (DICE) mediated by phiC31 and Bxb1 integrases was used to insert genes of interest flanked by phiC31 and Bxb1 attB sites at the H11 locus, replacing the landing pad. This system provided complete control over content, direction and copy number of inserted genes, with a specificity of 100%. A series of genes, including mCherry and various combinations of the neural transcription factors LMX1a, FOXA2 and OTX2, were inserted in recipient cell lines derived from H9 ESC, as well as iPSC lines derived from a Parkinson’s disease patient and a normal sibling control. The DICE system offers rapid, efficient and precise gene insertion in ESC and iPSC and is particularly well suited for repeated modifications of the same locus.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 30
    Publication Date: 2014-03-13
    Description: Recombineering, which is the use of homologous recombination for DNA engineering in Escherichia coli , usually uses antibiotic selection to identify the intended recombinant. When combined in a second step with counterselection using a small molecule toxin, seamless products can be obtained. Here, we report the advantages of a genetic strategy using CcdB as the counterselectable agent. Expression of CcdB is toxic to E. coli in the absence of the CcdA antidote so counterselection is initiated by the removal of CcdA expression. CcdB counterselection is robust and does not require titrations or experiment-to-experiment optimization. Because counterselection strategies necessarily differ according to the copy number of the target, we describe two variations. For multi-copy targets, we use two E. coli hosts so that counterselection is exerted by the transformation step that is needed to separate the recombined and unrecombined plasmids. For single copy targets, we put the ccdA gene onto the temperature-sensitive pSC101 Red expression plasmid so that counterselection is exerted by the standard temperature shift to remove the expression plasmid. To reduce unwanted intramolecular recombination, we also combined CcdB counterselection with Redα omission. These options improve the use of counterselection in recombineering with BACs, plasmids and the E. coli chromosome.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 31
    Publication Date: 2013-12-07
    Description: The two-step process of selection and counter-selection is a standard way to enable genetic modification and engineering of bacterial genomes using homologous recombination methods. The tetA and sacB genes are contained in a DNA cassette and confer a novel dual counter-selection system. Expression of tetA confers bacterial resistance to tetracycline (Tc R ) and also causes sensitivity to the lipophillic chelator fusaric acid; sacB causes sensitivity to sucrose. These two genes are introduced as a joint DNA cassette into Escherichia coli by selection for Tc R . A medium containing both fusaric acid and sucrose has been developed, in which, coexpression of tetA-sacB is orders of magnitude more sensitive as a counter-selection agent than either gene alone. In conjunction with the homologous recombination methods of recombineering and P1 transduction, this powerful system has been used to select changes in the bacterial genome that cannot be directly detected by other counter-selection systems.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 32
    Publication Date: 2014-02-28
    Description: DNA ‘assembly’ from ‘building blocks’ remains a cornerstone in synthetic biology, whether it be for gene synthesis (~1 kb), pathway engineering (~10 kb) or synthetic genomes (〉100 kb). Despite numerous advances in the techniques used for DNA assembly, verification of the assembly is still a necessity, which becomes cost-prohibitive and a logistical challenge with increasing scale. Here we describe for the first time a comprehensive, high-throughput solution for structural DNA assembly verification by restriction digest using exhaustive in silico enzyme screening, rolling circle amplification of plasmid DNA, capillary electrophoresis and automated digest pattern recognition. This low-cost and robust methodology has been successfully used to screen over 31 000 clones of DNA constructs at 〈$1 per sample.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 33
    Publication Date: 2014-02-28
    Description: Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by C31 integrase. Using six orthogonal attP / attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. C31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 34
    Publication Date: 2014-04-03
    Description: A conditional gene expression system that is fast-acting, is tunable and achieves single-gene specificity was recently developed for yeast. A gene placed directly downstream of a modified GAL1 promoter containing six Zif268 binding sequences (with single nucleotide spacing) was shown to be selectively inducible in the presence of β-estradiol, so long as cells express the artificial transcription factor, Z 3 EV (a fusion of the Zif268 DNA binding domain, the ligand binding domain of the human estrogen receptor and viral protein 16). We show the strength of Z 3 EV-responsive promoters can be modified using straightforward design principles. By moving Zif268 binding sites toward the transcription start site, expression output can be nearly doubled. Despite the reported requirement of estrogen receptor dimerization for hormone-dependent activation, a single binding site suffices for target gene activation. Target gene expression levels correlate with promoter binding site copy number and we engineer a set of inducible promoter chassis with different input–output characteristics. Finally, the coupling between inducer identity and gene activation is flexible: the ligand specificity of Z 3 EV can be re-programmed to respond to a non-hormone small molecule with only five amino acid substitutions in the human estrogen receptor domain, which may prove useful for industrial applications.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 35
    Publication Date: 2012-03-29
    Description: We demonstrate a system for cloning and modifying the chloroplast genome from the green alga, Chlamydomonas reinhardtii . Through extensive use of sequence stabilization strategies, the ex vivo genome is assembled in yeast from a collection of overlapping fragments. The assembled genome is then moved into bacteria for large-scale preparations and transformed into C. reinhardtii cells. This system also allows for the generation of simultaneous, systematic and complex genetic modifications at multiple loci in vivo. We use this system to substitute genes encoding core subunits of the photosynthetic apparatus with orthologs from a related alga, Scenedesmus obliquus . Once transformed into algae, the substituted genome recombines with the endogenous genome, resulting in a hybrid plastome comprising modifications in disparate loci. The in vivo function of the genomes described herein demonstrates that simultaneous engineering of multiple sites within the chloroplast genome is now possible. This work represents the first steps toward a novel approach for creating genetic diversity in any or all regions of a chloroplast genome.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 36
    Publication Date: 2012-02-17
    Description: The increasing interest in genetic manipulation of bacterial host metabolic pathways for protein or small molecule production has led to a need to add new genes to a chromosome quickly and easily without leaving behind a selectable marker. The present report describes a vector and four-day procedure that enable site-specific chromosomal insertion of cloned genes in a context insulated from external transcription, usable once in a construction series. The use of rhamnose-inducible transcription from rhaBp allows regulation of the inserted genes independently of the commonly used IPTG and arabinose strategies. Using lacZ as a reporter, we first show that expression from the rhamnose promoter is tightly regulatable, exhibiting very low leakage of background expression compared with background, and moderate rhamnose-induced expression compared with IPTG-induced expression from lacp . Second, the expression of a DNA methyltransferase was used to show that rhamnose regulation yielded on-off expression of this enzyme, such that a resident high-copy plasmid was either fully sensitive or fully resistant to isoschizomer restriction enzyme cleavage. In both cases, growth medium manipulation allows intermediate levels of expression. The vehicle can also be adapted as an ORF-cloning vector.
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  • 37
    Publication Date: 2012-02-17
    Description: The development of economical and high-throughput gene synthesis technology has been hampered by the high occurrence of errors in the synthesized products, which requires expensive labor and time to correct. Here, we describe an error correction reaction (ECR), which employs Surveyor, a mismatch-specific DNA endonuclease, to remove errors from synthetic genes. In ECR reactions, errors are revealed as mismatches by re-annealing of the synthetic gene products. Mismatches are recognized and excised by a combination of mismatch-specific endonuclease and 3'-〉5' exonuclease activities in the reaction mixture. Finally, overlap extension polymerase chain reaction (OE-PCR) re-assembles the resulting fragments into intact genes. The process can be iterated for increased fidelity. With two iterations, we were able to reduce errors in synthetic genes by 〉16-fold, yielding a final error rate of ~1 in 8700 bp.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 38
    Publication Date: 2014-09-27
    Description: The precise control of gene expression is essential in basic biological research as well as in biotechnological applications. Most regulated systems available in yeast enable only the overexpression of the target gene, excluding the possibility of intermediate or weak expression. Moreover, these systems are frequently toxic or depend on growth conditions. We constructed a heterologous transcription factor that overcomes these limitations. Our system is a fusion of the bacterial LexA DNA-binding protein, the human estrogen receptor (ER) and an activation domain (AD). The activity of this chimera, called LexA-ER-AD, is tightly regulated by the hormone β-estradiol. The selection of the AD proved to be crucial to avoid toxic effects and to define the range of activity that can be precisely tuned with β-estradiol. As our system is based on a heterologous DNA-binding domain, induction in different metabolic contexts is possible. Additionally, by controlling the number of LexA-binding sites in the target promoter, one can scale the expression levels up or down. Overall, our LexA-ER-AD system is a valuable tool to precisely control gene expression in different experimental contexts without toxic side effects.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 39
    Publication Date: 2014-09-27
    Description: Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bioproducts, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 40
    Publication Date: 2014-11-28
    Description: Mammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To address this problem, here we present a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units, 27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept, we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements, genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 41
    Publication Date: 2012-10-10
    Description: A major challenge in metabolic engineering and synthetic biology is to balance the flux of an engineered heterologous metabolic pathway to achieve high yield and productivity in a target organism. Here, we report a simple, efficient and programmable approach named ‘customized optimization of metabolic pathways by combinatorial transcriptional engineering (COMPACTER)’ for rapid tuning of gene expression in a heterologous pathway under distinct metabolic backgrounds. Specifically, a library of mutant pathways is created by de novo assembly of promoter mutants of varying strengths for each pathway gene in a target organism followed by high-throughput screening/selection. To demonstrate this approach, a single round of COMPACTER was used to generate both a xylose utilizing pathway with near-highest efficiency and a cellobiose utilizing pathway with highest efficiency that were ever reported in literature for both laboratory and industrial yeast strains. Interestingly, these engineered xylose and cellobiose utilizing pathways were all host-specific. Therefore, COMPACTER represents a powerful approach to tailor-make metabolic pathways for different strain backgrounds, which is difficult if not impossible to achieve by existing pathway engineering methods.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 42
    Publication Date: 2012-10-10
    Description: We developed a highly scalable ‘shotgun’ DNA synthesis technology by utilizing microchip oligonucleotides, shotgun assembly and next-generation sequencing technology. A pool of microchip oligonucleotides targeting a penicillin biosynthetic gene cluster were assembled into numerous random fragments, and tagged with 20 bp degenerate barcode primer pairs. An optimal set of error-free fragments were identified by high-throughput DNA sequencing, selectively amplified using the barcode sequences, and successfully assembled into the target gene cluster.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 43
    Publication Date: 2013-09-06
    Description: We developed a framework for quick and reliable construction of complex gene circuits for genetically engineering mammalian cells. Our hierarchical framework is based on a novel nucleotide addressing system for defining the position of each part in an overall circuit. With this framework, we demonstrate construction of synthetic gene circuits of up to 64 kb in size comprising 11 transcription units and 33 basic parts. We show robust gene expression control of multiple transcription units by small molecule inducers in human cells with transient transfection and stable chromosomal integration of these circuits. This framework enables development of complex gene circuits for engineering mammalian cells with unprecedented speed, reliability and scalability and should have broad applicability in a variety of areas including mammalian cell fermentation, cell fate reprogramming and cell-based assays.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 44
    Publication Date: 2014-04-15
    Description: RGB marking and DNA barcoding are two cutting-edge technologies in the field of clonal cell marking. To combine the virtues of both approaches, we equipped LeGO vectors encoding red, green or blue fluorescent proteins with complex DNA barcodes carrying color-specific signatures. For these vectors, we generated highly complex plasmid libraries that were used for the production of barcoded lentiviral vector particles. In proof-of-principle experiments, we used barcoded vectors for RGB marking of cell lines and primary murine hepatocytes. We applied single-cell polymerase chain reaction to decipher barcode signatures of individual RGB-marked cells expressing defined color hues. This enabled us to prove clonal identity of cells with one and the same RGB color. Also, we made use of barcoded vectors to investigate clonal development of leukemia induced by ectopic oncogene expression in murine hematopoietic cells. In conclusion, by combining RGB marking and DNA barcoding, we have established a novel technique for the unambiguous genetic marking of individual cells in the context of normal regeneration as well as malignant outgrowth. Moreover, the introduction of color-specific signatures in barcodes will facilitate studies on the impact of different variables (e.g. vector type, transgenes, culture conditions) in the context of competitive repopulation studies.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 45
    Publication Date: 2014-04-15
    Description: Insertional oncogene activation and aberrant splicing have proved to be major setbacks for retroviral stem cell gene therapy. Integrase-deficient human immunodeficiency virus-1-derived vectors provide a potentially safer approach, but their circular genomes are rapidly lost during cell division. Here we describe a novel lentiviral vector (LV) that incorporates human ß-interferon scaffold/matrix-associated region sequences to provide an origin of replication for long-term mitotic maintenance of the episomal LTR circles. The resulting ‘anchoring’ non-integrating lentiviral vector (aniLV) achieved initial transduction rates comparable with integrating vector followed by progressive establishment of long-term episomal expression in a subset of cells. Analysis of aniLV-transduced single cell-derived clones maintained without selective pressure for 〉100 rounds of cell division showed sustained transgene expression from episomes and provided molecular evidence for long-term episome maintenance. To evaluate aniLV performance in primary cells, we transduced lineage-depleted murine hematopoietic progenitor cells, observing GFP expression in clonogenic progenitor colonies and peripheral blood leukocyte chimerism following transplantation into conditioned hosts. In aggregate, our studies suggest that scaffold/matrix-associated region elements can serve as molecular anchors for non-integrating lentivector episomes, providing sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo .
    Keywords: Synthetic Biology and Assembly Cloning
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  • 46
    Publication Date: 2014-11-12
    Description: Assembly of DNA ‘parts’ to create larger constructs is an essential enabling technique for bioengineering and synthetic biology. Here we describe a simple method, PaperClip, which allows flexible assembly of multiple DNA parts from currently existing libraries cloned in any vector. No restriction enzymes, mutagenesis of internal restriction sites, or reamplification to add end homology are required. Order of assembly is directed by double stranded oligonucleotides—‘Clips’. Clips are formed by ligation of pairs of oligonucleotides corresponding to the ends of each part. PaperClip assembly can be performed by polymerase chain reaction or by cell extract-mediated recombination. Once multi-use Clips have been prepared, assembly of at least six DNA parts in any order can be accomplished with high efficiency within several hours.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 47
    Publication Date: 2014-08-15
    Description: Synthetic biology has significantly advanced the design of mammalian trigger-inducible transgene-control devices that are able to programme complex cellular behaviour. Fruit-based benzoate derivatives licensed as food additives, such as flavours (e.g. vanillate) and preservatives (e.g. benzoate), are a particularly attractive class of trigger compounds for orthogonal mammalian transgene control devices because of their innocuousness, physiological compatibility and simple oral administration. Capitalizing on the genetic componentry of the soil bacterium Comamonas testosteroni , which has evolved to catabolize a variety of aromatic compounds, we have designed different mammalian gene expression systems that could be induced and repressed by the food additives benzoate and vanillate. When implanting designer cells engineered for gene switch-driven expression of the human placental secreted alkaline phosphatase (SEAP) into mice, blood SEAP levels of treated animals directly correlated with a benzoate-enriched drinking programme. Additionally, the benzoate-/vanillate-responsive device was compatible with other transgene control systems and could be assembled into higher-order control networks providing expression dynamics reminiscent of a lap-timing stopwatch. Designer gene switches using licensed food additives as trigger compounds to achieve antagonistic dual-input expression profiles and provide novel control topologies and regulation dynamics may advance future gene- and cell-based therapies.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 48
    Publication Date: 2012-12-14
    Description: Multivalent molecular interactions can be exploited to dramatically enhance the performance of an affinity reagent. The enhancement in affinity and specificity achieved with a multivalent construct depends critically on the effectiveness of the scaffold that joins the ligands, as this determines their positions and orientations with respect to the target molecule. Currently, no generalizable design rules exist for construction of an optimal multivalent ligand for targets with known structures, and the design challenge remains an insurmountable obstacle for the large number of proteins whose structures are not known. As an alternative to such design-based strategies, we report here a directed evolution-based method for generating optimal bivalent aptamers. To demonstrate this approach, we fused two thrombin aptamers with a randomized DNA sequence and used a microfluidic in vitro selection strategy to isolate scaffolds with exceptionally high affinities. Within five rounds of selection, we generated a bivalent aptamer that binds thrombin with an apparent dissociation constant (K d ) 〈10 pM, representing a ~200-fold improvement in binding affinity over the monomeric aptamers and a ~15-fold improvement over the best designed bivalent construct. The process described here can be used to produce high-affinity multivalent aptamers and could potentially be adapted to other classes of biomolecules.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 49
    Publication Date: 2013-07-16
    Description: Synthetic biology has significantly advanced the design of synthetic control devices, gene circuits and networks that can reprogram mammalian cells in a trigger-inducible manner. Prokaryotic helix-turn-helix motifs have become the standard resource to design synthetic mammalian transcription factors that tune chimeric promoters in a small molecule-responsive manner. We have identified a family of Actinomycetes transcriptional repressor proteins showing a tandem TetR-family signature and have used a synthetic biology-inspired approach to reveal the potential control dynamics of these bi-partite regulators. Daisy-chain assembly of well-characterized prokaryotic repressor proteins such as TetR, ScbR, TtgR or VanR and fusion to either the Herpes simplex transactivation domain VP16 or the Krueppel-associated box domain (KRAB) of the human kox-1 gene resulted in synthetic bi- and even tri-partite mammalian transcription factors that could reversibly program their individual chimeric or hybrid promoters for trigger-adjustable transgene expression using tetracycline (TET), -butyrolactones, phloretin and vanillic acid. Detailed characterization of the bi-partite ScbR-TetR-VP16 (ST-TA) transcription factor revealed independent control of TET- and -butyrolactone-responsive promoters at high and double-pole double-throw (DPDT) relay switch qualities at low intracellular concentrations. Similar to electromagnetically operated mechanical DPDT relay switches that control two electric circuits by a fully isolated low-power signal, TET programs ST-TA to progressively switch from TetR-specific promoter-driven expression of transgene one to ScbR-specific promoter-driven transcription of transgene two while ST-TA flips back to exclusive transgene 1 expression in the absence of the trigger antibiotic. We suggest that natural repressors and activators with tandem TetR-family signatures may also provide independent as well as DPDT-mediated control of two sets of transgenes in bacteria, and that their synthetic transcription-factor analogs may enable the design of compact therapeutic gene circuits for gene and cell-based therapies.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 50
    Publication Date: 2012-08-08
    Description: Cy3B is an extremely bright and stable fluorescent dye, which is only available for coupling to nucleic acids post-synthetically. This severely limits its use in the fields of genomics, biology and nanotechnology. We have optimized the synthesis of Cy3B, and for the first time produced a diverse range of Cy3B monomers for use in solid-phase oligonucleotide synthesis. This molecular toolkit includes phosphoramidite monomers with Cy3B linked to deoxyribose, to the 5-position of thymine, and to a hexynyl linker, in addition to an oligonucleotide synthesis resin in which Cy3B is linked to deoxyribose. These monomers have been used to incorporate single and multiple Cy3B units into oligonucleotides internally and at both termini. Cy3B Taqman probes, Scorpions and HyBeacons have been synthesized and used successfully in mutation detection, and a dual Cy3B Molecular Beacon was synthesized and found to be superior to the corresponding Cy3B/DABCYL Beacon. Attachment of Cy3, Cy3B and Cy5 to the 5-position of thymidine by an ethynyl linker enabled the synthesis of an oligonucleotide FRET system. The rigid linker between the dye and nucleobase minimizes dye–dye and dye–DNA interactions and reduces fluorescence quenching. These reagents open up new future applications of Cy3B, including more sensitive single-molecule and cell-imaging studies.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 51
    Publication Date: 2013-01-20
    Description: Synthetic RNA control devices that use ribozymes as gene-regulatory components have been applied to controlling cellular behaviors in response to environmental signals. Quantitative measurement of the in vitro cleavage rate constants associated with ribozyme-based devices is essential for advancing the molecular design and optimization of this class of gene-regulatory devices. One of the key challenges encountered in ribozyme characterization is the efficient generation of full-length RNA from in vitro transcription reactions, where conditions generally lead to significant ribozyme cleavage. Current methods for generating full-length ribozyme-encoding RNA rely on a trans-blocking strategy, which requires a laborious gel separation and extraction step. Here, we develop a simple two-step gel-free process including cis-blocking and trans-activation steps to support scalable generation of functional full-length ribozyme-encoding RNA. We demonstrate our strategy on various types of natural ribozymes and synthetic ribozyme devices, and the cleavage rate constants obtained for the RNA generated from our strategy are comparable with those generated through traditional methods. We further develop a rapid, label-free ribozyme cleavage assay based on surface plasmon resonance, which allows continuous, real-time monitoring of ribozyme cleavage. The surface plasmon resonance-based characterization assay will complement the versatile cis-blocking and trans-activation strategy to broadly advance our ability to characterize and engineer ribozyme-based devices.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 52
    Publication Date: 2012-11-04
    Description: Recent advances have demonstrated the use of RNA-based control devices to program sophisticated cellular functions; however, the efficiency with which these devices can be quantitatively tailored has limited their broader implementation in cellular networks. Here, we developed a high-efficiency, high-throughput and quantitative two-color fluorescence-activated cell sorting-based screening strategy to support the rapid generation of ribozyme-based control devices with user-specified regulatory activities. The high-efficiency of this screening strategy enabled the isolation of a single functional sequence from a library of over 10 6 variants within two sorting cycles. We demonstrated the versatility of our approach by screening large libraries generated from randomizing individual components within the ribozyme device platform to efficiently isolate new device sequences that exhibit increased in vitro cleavage rates up to 10.5-fold and increased in vivo activation ratios up to 2-fold. We also identified a titratable window within which in vitro cleavage rates and in vivo gene-regulatory activities are correlated, supporting the importance of optimizing RNA device activity directly in the cellular environment. Our two-color fluorescence-activated cell sorting-based screen provides a generalizable strategy for quantitatively tailoring genetic control elements for broader integration within biological networks.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 53
    Publication Date: 2013-08-28
    Description: The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 54
    Publication Date: 2013-08-28
    Description: Antisense RNA transcription attenuators are a key component of the synthetic biology toolbox, with their ability to serve as building blocks for both signal integration logic circuits and transcriptional cascades. However, a central challenge to building more sophisticated RNA genetic circuitry is creating larger families of orthogonal attenuators that function independently of each other. Here, we overcome this challenge by developing a modular strategy to create chimeric fusions between the engineered transcriptional attenuator from plasmid pT181 and natural antisense RNA translational regulators. Using in vivo gene expression assays in Escherichia coli , we demonstrate our ability to create chimeric attenuators by fusing sequences from five different translational regulators. Mutagenesis of these functional attenuators allowed us to create a total of 11 new chimeric attenutaors. A comprehensive orthogonality test of these culminated in a 7 x 7 matrix of mutually orthogonal regulators. A comparison between all chimeras tested led to design principles that will facilitate further engineering of orthogonal RNA transcription regulators, and may help elucidate general principles of non-coding RNA regulation. We anticipate that our strategy will accelerate the development of even larger families of orthogonal RNA transcription regulators, and thus create breakthroughs in our ability to construct increasingly sophisticated RNA genetic circuitry.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 55
    Publication Date: 2013-04-23
    Description: Studying complex biological processes such as cancer development, stem cell induction and transdifferentiation requires the modulation of multiple genes or pathways at one time in a single cell. Herein, we describe straightforward methods for rapid and efficient assembly of bacterial marker free multigene cassettes containing up to six complementary DNAs/short hairpin RNAs. We have termed this method RecWay assembly, as it makes use of both Cre recombinase and the commercially available Gateway cloning system. Further, because RecWay assembly uses truly modular components, it allows for the generation of randomly assembled multigene vector libraries. These multigene vectors are integratable, and later excisable, using the highly efficient piggyBac ( PB ) DNA transposon system. Moreover, we have dramatically improved the expression of stably integrated multigene vectors by incorporation of insulator elements to prevent promoter interference seen with multigene vectors. We demonstrate that insulated multigene PB transposons can stably integrate and faithfully express up to five fluorescent proteins and the puromycin-thymidine kinase resistance gene in vitro , with up to 70-fold higher gene expression compared with analogous uninsulated vectors . RecWay assembly of multigene transposon vectors allows for widely applicable modelling of highly complex biological processes and can be easily performed by other research laboratories.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 56
    Publication Date: 2013-04-23
    Description: Techniques for assembly of designed DNA sequences are important for synthetic biology. So far, a few methods have been developed towards high-throughput seamless DNA assembly in vitro , including both the homologous sequences-based system and the type IIS-mediated system. Here, we describe a novel method designated ‘MASTER Ligation’, by which multiple DNA sequences can be seamlessly assembled through a simple and sequence-independent hierarchical procedure. The key restriction endonuclease used, MspJI, shares both type IIM and type IIS properties; thus, it only recognizes the methylation-specific 4-bp sites, m CNNR (R = A or G), and cuts DNA outside of the recognition sequences. This method was tested via successful assembly of either multiple polymerase chain reaction amplicons or restriction fragments of the actinorhodin biosynthetic cluster of Streptomyces coelicolor (~29 kb), which was further heterologously expressed in a fast-growing and moderately thermophilic strain, Streptomyces sp. 4F.
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  • 57
    Publication Date: 2013-02-20
    Description: Bacterial operons are nature’s tool for regulating and coordinating multi-gene expression in prokaryotes. They are also a gene architecture commonly used in the biosynthesis of many pharmaceutically important compounds and industrially useful chemicals. Despite being an important eukaryotic production host, Saccharomyces cerevisiae has never had such gene architecture. Here, we report the development of a system to assemble and regulate a multi-gene pathway in S. cerevisiae . Full pathways can be constructed using pre-made parts from a plasmid toolbox. Subsequently, through the use of a yeast strain containing a stably integrated gene switch, the assembled pathway can be regulated using a readily available and inexpensive compound—estradiol—with extremely high sensitivity (10 nM). To demonstrate the use of the system, we assembled the five-gene zeaxanthin biosynthetic pathway in a single step and showed the ligand-dependent coordinated expression of all five genes as well as the tightly regulated production of zeaxanthin. Compared with a previously reported constitutive zeaxanthin pathway, our inducible pathway was shown to have 50-fold higher production level.
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  • 58
    Publication Date: 2013-02-20
    Description: Zinc-finger nucleases (ZFNs) have been used for genome engineering in a wide variety of organisms; however, it remains challenging to design effective ZFNs for many genomic sequences using publicly available zinc-finger modules. This limitation is in part because of potential finger–finger incompatibility generated on assembly of modules into zinc-finger arrays (ZFAs). Herein, we describe the validation of a new set of two-finger modules that can be used for building ZFAs via conventional assembly methods or a new strategy—finger stitching—that increases the diversity of genomic sequences targetable by ZFNs. Instead of assembling ZFAs based on units of the zinc-finger structural domain, our finger stitching method uses units that span the finger–finger interface to ensure compatibility of neighbouring recognition helices. We tested this approach by generating and characterizing eight ZFAs, and we found their DNA-binding specificities reflected the specificities of the component modules used in their construction. Four pairs of ZFNs incorporating these ZFAs generated targeted lesions in vivo , demonstrating that stitching yields ZFAs with robust recognition properties.
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  • 59
    Publication Date: 2013-05-04
    Description: The reliable forward engineering of genetic systems remains limited by the ad hoc reuse of many types of basic genetic elements. Although a few intrinsic prokaryotic transcription terminators are used routinely, termination efficiencies have not been studied systematically. Here, we developed and validated a genetic architecture that enables reliable measurement of termination efficiencies. We then assembled a collection of 61 natural and synthetic terminators that collectively encode termination efficiencies across an ~800-fold dynamic range within Escherichia coli . We simulated co-transcriptional RNA folding dynamics to identify competing secondary structures that might interfere with terminator folding kinetics or impact termination activity. We found that structures extending beyond the core terminator stem are likely to increase terminator activity. By excluding terminators encoding such context-confounding elements, we were able to develop a linear sequence-function model that can be used to estimate termination efficiencies ( r = 0.9, n = 31) better than models trained on all terminators ( r = 0.67, n = 54). The resulting systematically measured collection of terminators should improve the engineering of synthetic genetic systems and also advance quantitative modeling of transcription termination.
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  • 60
    Publication Date: 2013-06-28
    Description: Recombineering in bacteria is a powerful technique for genome reconstruction, but until now, it was not generally applicable for development of small-molecule producers because of the inconspicuous phenotype of most compounds of biotechnological relevance. Here, we establish recombineering for Corynebacterium glutamicum using RecT of prophage Rac and combine this with our recently developed nanosensor technology, which enables the detection and isolation of productive mutants at the single-cell level via fluorescence-activated cell sorting (FACS). We call this new technology RecFACS, which we use for genomic site-directed saturation mutagenesis without relying on pre-constructed libraries to directly isolate l -lysine-producing cells. A mixture of 19 different oligonucleotides was used targeting codon 81 in murE of the wild-type, at a locus where one single mutation is known to cause l -lysine production. Using RecFACS, productive mutants were screened and isolated. Sequencing revealed 12 different amino acid exchanges in the targeted murE codon, which caused different l -lysine production titers. Apart from introducing a rapid genome construction technology for C. glutamicum , the present work demonstrates that RecFACS is suitable to simply create producers as well as genetic diversity in one single step, thus establishing a new general concept in synthetic biology.
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  • 61
    Publication Date: 2013-11-02
    Description: The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5' coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11 , were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.
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  • 62
    Publication Date: 2013-11-02
    Description: Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR–Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR–CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion. This allows customized changes in the C. elegans genome by homologous recombination: sequences contained in the repair template (the transgene) are copied by gene conversion into the genome. The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 63
    Publication Date: 2013-11-02
    Description: The generation of genome-modified animals is a powerful approach to analyze gene functions. The CAS9/guide RNA (gRNA) system is expected to become widely used for the efficient generation of genome-modified animals, but detailed studies on optimum conditions and availability are limited. In the present study, we attempted to generate large-scale genome-modified mice with an optimized CAS9/gRNA system, and confirmed the transmission of these mutations to the next generations. A comparison of different types of gRNA indicated that the target loci of almost all pups were modified successfully by the use of long-type gRNAs with CAS9. We showed that this system has much higher mutation efficiency and much lower off-target effect compared to zinc-finger nuclease. We propose that most of these off-target effects can be avoided by the careful control of CAS9 mRNA concentration and that the genome-modification efficiency depends rather on the gRNA concentration. Under optimized conditions, large-scale (~10 kb) genome-modified mice can be efficiently generated by modifying two loci on a single chromosome using two gRNAs at once in mouse zygotes. In addition, the normal transmission of these CAS9/gRNA-induced mutations to the next generation was confirmed. These results indicate that CAS9/gRNA system can become a highly effective tool for the generation of genome-modified animals.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 64
    Publication Date: 2013-04-14
    Description: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems in bacteria and archaea use RNA-guided nuclease activity to provide adaptive immunity against invading foreign nucleic acids. Here, we report the use of type II bacterial CRISPR-Cas system in Saccharomyces cerevisiae for genome engineering. The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. Using constitutive Cas9 expression and a transient gRNA cassette, we show that targeted double-strand breaks can increase homologous recombination rates of single- and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. In addition, co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. Our approach provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 65
    Publication Date: 2013-08-09
    Description: Customized TALENs and Cas9/gRNAs have been used for targeted mutagenesis in zebrafish to induce indels into protein-coding genes. However, indels are usually not sufficient to disrupt the function of non-coding genes, gene clusters or regulatory sequences, whereas large genomic deletions or inversions are more desirable for this purpose. By injecting two pairs of TALEN mRNAs or two gRNAs together with Cas9 mRNA targeting distal DNA sites of the same chromosome, we obtained predictable genomic deletions or inversions with sizes ranging from several hundred bases to nearly 1 Mb. We have successfully achieved this type of modifications for 11 chromosomal loci by TALENs and 2 by Cas9/gRNAs with different combinations of gRNA pairs, including clusters of miRNA and protein-coding genes. Seven of eight TALEN-targeted lines transmitted the deletions and one transmitted the inversion through germ line. Our findings indicate that both TALENs and Cas9/gRNAs can be used as an efficient tool to engineer genomes to achieve large deletions or inversions, including fragments covering multiple genes and non-coding sequences. To facilitate the analyses and application of existing ZFN, TALEN and CRISPR/Cas data, we have updated our EENdb database to provide a chromosomal view of all reported engineered endonucleases targeting human and zebrafish genomes.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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