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Schmerer M.,University of Texas at Austin | Molineux I.J.,University of Texas at Austin | Ally D.,Bayer Crop Science Biologics | Tyerman J.,Total New Energies United States Inc. | And 2 more authors.
Journal of Biological Engineering | Year: 2014

Background: In prior work, a phage engineered with a biofilm-degrading enzyme (dispersin B) cleared artificial, short-term biofilms more fully than the phage lacking the enzyme. An unresolved question is whether the transgene will be lost or maintained during phage growth - its loss would limit the utility of the engineering. Broadly supported evolutionary theory suggests that transgenes will be lost through a 'tragedy of the commons' mechanism unless the ecology of growth in biofilms meets specific requirements. We test that theory here. Results: Functional properties of the transgenic phage were identified. Consistent with the previous study, the dispersin phage was superior to unmodified phage at clearing short term biofilms grown in broth, shown here to be an effect attributable to free enzyme. However, the dispersin phage was only marginally better than control phages on short term biofilms in minimal media and was no better than control phages in clearing long term biofilms. There was little empirical support for the tragedy of the commons framework despite a strong theoretical foundation for its supposed relevance. The framework requires that the transgene imposes an intrinsic cost, yet the transgene was intrinsically neutral or beneficial when expressed from one part of the phage genome. Expressed from a different part of the genome, the transgene did behave as if intrinsically costly, but its maintenance did not benefit from spatially structured growth per se - violating the tragedy framework. Conclusions: Overall, the transgene was beneficial under many conditions, but no insight to its maintenance was attributable to the established evolutionary framework. The failure likely resides in system details that would be used to parameterize the models. Our study cautions against naive applications of evolutionary theory to synthetic biology, even qualitatively. © Schmerer et al.; licensee BioMed Central Ltd. Source

Chubukov V.,Joint BioEnergy Institute | Chubukov V.,Lawrence Berkeley National Laboratory | Mingardon F.,Total New Energies United States Inc. | Schackwitz W.,Joint Genome Institute | And 14 more authors.
Applied and Environmental Microbiology | Year: 2015

Limonene, a major component of citrus peel oil, has a number of applications related to microbiology. The antimicrobial properties of limonene make it a popular disinfectant and food preservative, while its potential as a biofuel component has made it the target of renewable production efforts through microbial metabolic engineering. For both applications, an understanding of microbial sensitivity or tolerance to limonene is crucial, but the mechanism of limonene toxicity remains enigmatic. In this study, we characterized a limonene-tolerant strain of Escherichia coli and found a mutation in ahpC, encoding alkyl hydroperoxidase, which alleviated limonene toxicity. We show that the acute toxicity previously attributed to limonene is largely due to the common oxidation product limonene hydroperoxide, which forms spontaneously in aerobic environments. The mutant AhpC protein with an L-to-Q change at position 177 (AhpCL177Q) was able to alleviate this toxicity by reducing the hydroperoxide to a more benign compound. We show that the degree of limonene toxicity is a function of its oxidation level and that nonoxidized limonene has relatively little toxicity to wild-type E. coli cells. Our results have implications for both the renewable production of limonene and the applications of limonene as an antimicrobial. © 2015, American Society for Microbiology. Source

Thakker C.,Rice University | Thakker C.,Total New Energies United States Inc. | Lin K.,Rice University | Martini-Stoica H.,Rice University | And 2 more authors.
Biotechnology Journal | Year: 2016

Various methods have been developed for gene disruption in bacteria; however, extra in vitro manipulation steps or the residual presence of a scar in the host chromosome limits the use of such methods. By utilizing the unique properties of ISHp608, we have developed a simple and precise method for genome manipulation in Escherichia coli that alters the gene sequence without leaving foreign DNA in the chromosome. This strategy involves PCR amplification of a DNA cassette containing ISHp608-LE (left end)-antibiotic resistance gene-counterselection marker-ISHp608-RE (right end) by using primers containing extensions homologous to the adjacent regions of the target gene on the chromosome. The λ Red mediated recombination of the PCR product and antibiotic resistance screening results in transformants with a modified gene target. The ISHp608-LE-antibiotic resistance gene-counterselection marker-ISHp608-RE cassette can then be excised using a temperature sensitive plasmid expressing the TnpA transposase, which precisely cleaves ISHp608-LE and ISHp608-RE without leaving a scar sequence. We demonstrated lacZ gene point mutation repair, two precise disruptions of the lacZ gene and constructed a library of lacZ variants having variable β-galactosidase activity by changing its ribosome binding site sequences using the ISHp608 system. This technique can be used in E. coli genome modification and could be extended for use in other bacteria. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Gogolin R.,Institute for Solar Energy Research Hamelin | Harder N.P.,Institute for Solar Energy Research Hamelin | Harder N.P.,Leibniz University of Hanover | Harder N.P.,Total New Energies United States Inc.
Journal of Applied Physics | Year: 2013

We investigate the correlation between increased apparent carrier lifetime in photoconductance-based lifetime measurements and actually reduced recombination lifetime as measured by photoluminescence measurements. These findings are further reconfirmed by I-V curve measurements of solar cells. In particular, we show experimental results for lifetime samples and solar cells with and without hydrogen passivation. In the samples and solar cells without hydrogen passivation, we find both a stronger trapping behavior and a lower recombination lifetime. Our model provides a consistent description of the observation of both, the increased apparent lifetime from carrier trapping and the decreasing recombination lifetime. In our model, both are caused by a single physical mechanism; i.e., by Recombination-Active-Trap (RAT) states. Upon fitting the experimental lifetime data, we find that the RAT-defect parameters for the hydrogen-passivated and non-hydrogen-passivated lifetime samples and solar cells are identical except for the defect concentration: hydrogen-passivation reduced the defect density by 50% in both, the lifetime samples and solar cells. We conclude that trapping should be considered as an indication for hidden, yet potentially strongly increased, low injection recombination activity. © 2013 AIP Publishing LLC. Source

Shapland E.B.,Amyris Biotechnologies Inc. | Holmes V.,Amyris Biotechnologies Inc. | Reeves C.D.,Amyris Biotechnologies Inc. | Sorokin E.,Amyris Biotechnologies Inc. | And 8 more authors.
ACS Synthetic Biology | Year: 2015

In recent years, next-generation sequencing (NGS) technology has greatly reduced the cost of sequencing whole genomes, whereas the cost of sequence verification of plasmids via Sanger sequencing has remained high. Consequently, industrial-scale strain engineers either limit the number of designs or take short cuts in quality control. Here, we show that over 4000 plasmids can be completely sequenced in one Illumina MiSeq run for less than $3 each (15X coverage), which is a 20-fold reduction over using Sanger sequencing (2X coverage). We reduced the volume of the Nextera tagmentation reaction by 100-fold and developed an automated workflow to prepare thousands of samples for sequencing. We also developed software to track the samples and associated sequence data and to rapidly identify correctly assembled constructs having the fewest defects. As DNA synthesis and assembly become a centralized commodity, this NGS quality control (QC) process will be essential to groups operating high-throughput pipelines for DNA construction. (Figure Presented). © 2015 American Chemical Society. Source

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