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Cambray G.,BIOFAB International Open Facility Advancing Biotechnology BIOFAB | Cambray G.,University of California at Berkeley | Guimaraes J.C.,BIOFAB International Open Facility Advancing Biotechnology BIOFAB | Guimaraes J.C.,University of California at Berkeley | And 15 more authors.
Nucleic Acids Research | Year: 2013

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. © 2013 The Author(s).


Goldbeck C.P.,Lawrence Berkeley National Laboratory | Jensen H.M.,Lawrence Berkeley National Laboratory | Jensen H.M.,University of California at Berkeley | Teravest M.A.,Cornell University | And 9 more authors.
ACS Synthetic Biology | Year: 2013

Introduction of the electron transfer complex MtrCAB from Shewanella oneidensis MR-1 into a heterologous host provides a modular and molecularly defined route for electrons to be transferred to an extracellular inorganic solid. However, an Escherichia coli strain expressing this pathway displayed limited control of MtrCAB expression and impaired cell growth. To overcome these limitations and to improve heterologous extracellular electron transfer, we used an E. coli host with a more tunable induction system and a panel of constitutive promoters to generate a library of strains that separately transcribe the mtr and cytochrome c maturation (ccm) operons over 3 orders of magnitude. From this library, we identified strains that show 2.2 times higher levels of MtrC and MtrA and that have improved cell growth. We find that a ∼300-fold decrease in the efficiency of MtrC and MtrA synthesis with increasing mtr promoter activity critically limits the maximum expression level of MtrC and MtrA. We also tested the extracellular electron transfer capabilities of a subset of the strains using a three-electrode microbial electrochemical system. Interestingly, the strain with improved cell growth and fewer morphological changes generated the largest maximal current per cfu, rather than the strain with more MtrC and MtrA. This strain also showed ∼30-fold greater maximal current per cfu than its ccm-only control strain. Thus, the conditions for optimal MtrCAB expression and anode reduction are distinct, and minimal perturbations to cell morphology are correlated with improved extracellular electron transfer in E. coli. © 2013 American Chemical Society.


PubMed | BIOFAB International Open Facility Advancing Biotechnology BIOFAB
Type: Journal Article | Journal: Nucleic acids research | Year: 2013

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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