Using Synthetic Biological Parts and Microbioreactors to Explore the Protein Expression Characteristics of <i>Escherichia coli</i>

Gorochowski, Thomas E.; Berg, Eric van den; Kerkman, Richard; Roubos, Johannes A.; Bovenberg, Roel A. L.

doi:10.1021/sb4001245

Cited by 40 publications

(31 citation statements)

References 43 publications

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“…RBSs function in either, because the mRNA-binding region of the 16S rRNA is identical, T7 RNAP functions in both and the plasmid origins can be carried in either. However, the quantitative function of these parts could be different owing to changes in the cellular environment, growth rate, metabolism and concentration of RNAPs and ribosomes 36,37 . This could lead to differences in the selection of parts to optimize activity in each organism.…”

Section: Transfer Of the Refactored Gene Cluster To E Colimentioning

confidence: 99%

Functional optimization of gene clusters by combinatorial design and assembly

et al. 2014

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Large microbial gene clusters encode useful functions, including energy utilization and natural product biosynthesis, but genetic manipulation of such systems is slow, difficult and complicated by complex regulation. We exploit the modularity of a refactored Klebsiella oxytoca nitrogen fixation (nif) gene cluster (16 genes, 103 parts) to build genetic permutations that could not be achieved by starting from the wild-type cluster. Constraint-based combinatorial design and DNA assembly are used to build libraries of radically different cluster architectures by varying part choice, gene order, gene orientation and operon occupancy. We construct 84 variants of the nifUSVWZM operon, 145 variants of the nifHDKY operon, 155 variants of the nifHDKYENJ operon and 122 variants of the complete 16-gene pathway. The performance and behavior of these variants are characterized by nitrogenase assay and strand-specific RNA sequencing (RNA-seq), and the results are incorporated into subsequent design cycles. We have produced a fully synthetic cluster that recovers 57% of wild-type activity. Our approach allows the performance of genetic parts to be quantified simultaneously in hundreds of genetic contexts. This parallelized design-build-test-learn cycle, which can access previously unattainable regions of genetic space, should provide a useful, fast tool for genetic optimization and hypothesis testing.

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Section: Transfer Of the Refactored Gene Cluster To E Colimentioning

confidence: 99%

Functional optimization of gene clusters by combinatorial design and assembly

et al. 2014

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“…A major challenge is predicting how a part will behave when assembled with many others (Cardinale et al , ). The sequences of surrounding parts (Poole et al , ), interactions with other circuit components or the host cell (Cardinale et al , ; Ceroni et al , ; Gyorgy et al , ; Gorochowski et al , ), and the general physiological state of the cell (Wohlgemuth et al , ; Gorochowski et al , ) can all alter a part's behavior. Although biophysical models have been refined to capture some contextual effects (Salis et al , ; Seo et al , ; Espah Borujeni et al , ), and new types of part created to insulate against these factors (Moon et al , ; Daniel et al , ; Mutalik et al , ; Siuti et al , ; Yang et al , ; Shendure et al , ), we have yet to reach a point where large and robust genetic circuits can be reliably built on our first attempt.…”

Section: Introductionmentioning

confidence: 99%

Absolute quantification of translational regulation and burden using combined sequencing approaches

Gorochowski

Chelysheva

Eriksen

et al. 2019

Molecular Systems Biology

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Translation of mRNA s into proteins is a key cellular process. Ribosome binding sites and stop codons provide signals to initiate and terminate translation, while stable secondary mRNA structures can induce translational recoding events. Fluorescent proteins are commonly used to characterize such elements but require the modification of a part's natural context and allow only a few parameters to be monitored concurrently. Here, we combine Ribo‐seq with quantitative RNA ‐seq to measure at nucleotide resolution and in absolute units the performance of elements controlling transcriptional and translational processes during protein synthesis. We simultaneously measure 779 translation initiation rates and 750 translation termination efficiencies across the Escherichia coli transcriptome, in addition to translational frameshifting induced at a stable RNA pseudoknot structure. By analyzing the transcriptional and translational response, we discover that sequestered ribosomes at the pseudoknot contribute to a σ 32 ‐mediated stress response, codon‐specific pausing, and a drop in translation initiation rates across the cell. Our work demonstrates the power of integrating global approaches toward a comprehensive and quantitative understanding of gene regulation and burden in living cells.

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“…If recombinant probiotic bacterial strains could colonize the mammalian gut and function for extended periods the potential for translation of such technologies to humans could be vastly improved 1 . Unfortunately, the burden that synthetic genetic circuits place on their bacterial hosts 3 can result in compensatory genetic mutation 4 , loss of engineered function 5 , or lack of growth of the recombinant strain 3 in a host and environment-dependent manner 6, 7 .…”

mentioning

confidence: 99%

Engineered bacteria can function in the mammalian gut long-term as live diagnostics of inflammation

et al. 2017

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Bacteria can be engineered to function as diagnostics or therapeutics in the mammalian gut but commercial translation of these technologies has been hindered by the susceptibility of synthetic genetic circuits to mutation and unpredictable function during extended gut colonization. Here we report stable, engineered bacterial strains that maintain their function for 6 months in the mouse gut. We engineered a commensal murine Escherichia coli strain to detect tetrathionate, which is produced during inflammation. Using our engineered diagnostic strain, which retains memory of exposure in the gut for analysis by fecal testing, we detected tetrathionate in both infection-induced and genetic mouse models of inflammation over 6 months. The synthetic genetic circuits in the engineered strain were genetically stable and functioned as intended over time. The robust, durable performance of these strains confirms the potential of engineered bacteria as living diagnostics.

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Using Synthetic Biological Parts and Microbioreactors to Explore the Protein Expression Characteristics of Escherichia coli

Cited by 40 publications

References 43 publications

Functional optimization of gene clusters by combinatorial design and assembly

Functional optimization of gene clusters by combinatorial design and assembly

Absolute quantification of translational regulation and burden using combined sequencing approaches

Engineered bacteria can function in the mammalian gut long-term as live diagnostics of inflammation

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