Genetically Programmable Microbial Assembly

Kozlowski, Mark T.; Silverman, Bradley; Johnstone, Christopher P.; Tirrell, David A.

doi:10.1021/acssynbio.0c00616

Cited by 13 publications

(6 citation statements)

References 47 publications

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“…The secretion signal sequences of estA and estB were excluded. The gene sequences of mWasabi and APEX2 were codon-optimized (IDT Codon Optimization Tool) based on their protein sequences. , Gene sequences of T7 RNAP and the T7 promoter, along with the lac operator and hammerhead ribozyme, were amplified from a previously reported construct (GenBank MN005204) . The endogenous promoters, P cotG , P cotZ , and P cotV , were amplified from the genome of B.…”

Section: Methodsmentioning

confidence: 99%

Stress-Tolerant, Recyclable, and Renewable Biocatalyst Platform Enabled by Engineered Bacterial Spores

2022

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Here, we describe a stress-tolerant, recyclable, and renewable biocatalyst platform based on T7 RNA polymerase-enabled high-density protein display on bacterial spores (TIED). TIED uses high-level T7 RNA polymerase-driven expression of recombinant proteins specifically in sporulating cells to allow spontaneous assembly of recombinant fusion proteins on the Bacillus subtilis spore surface. TIED enables high loading density in the range of 106 to 107 recombinant enzymes per spore, robust catalytic activity of displayed enzymes comparable to the respective free enzymes, and enhanced kinetic stability of displayed enzymes in methanol and elevated temperatures. Furthermore, we demonstrate TIED enzymes to be not only recyclable but also fully renewable after the loss of activity through induction of germination and sporulation, enabling perpetual regeneration of these immobilized biocatalysts.

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Section: Methodsmentioning

confidence: 99%

Stress-Tolerant, Recyclable, and Renewable Biocatalyst Platform Enabled by Engineered Bacterial Spores

2022

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“…Gene sequences of mWasabi and APEX2 were codon-optimized (IDT Codon Optimization Tool) based on their protein sequences. 42,46 Gene sequences of T7 RNAP and the T7 promoter, along with the lac operator and hammered Ribozyme were amplified from a previously reported construct (GenBank MN005204). 47 The endogenous promoters, P cotG , P cotZ , and P cotV , were amplified from the genome of B. subtilis PY79.…”

Section: Methodsmentioning

confidence: 99%

Stress-tolerant, recyclable, and autonomously renewable biocatalyst platform enabled by engineered bacterial spores

Hui

Cui

Sim

2022

Preprint

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Here, we describe a stress-tolerant, recyclable, and autonomously renewable biocatalyst platform based on T7 RNA polymerase-enabled high-density protein display on bacterial spores (TIED). TIED uses high-level T7 RNA polymerase-driven expression of recombinant proteins specifically in sporulating cells to allow spontaneous assembly of recombinant fusion proteins on B. subtilis spore surface. TIED enables a high loading density in the range of 106–107 recombinant enzymes per spore, robust catalytic activities of displayed enzymes comparable to the respective free enzymes, and enhanced kinetic stability of displayed enzymes in methanol and elevated temperatures. Further, we demonstrate TIED-enzymes to be not only recyclable, but fully renewable after loss of activity through induction of germination and sporulation, enabling perpetual reuse of these immobilized biocatalysts.Graphical abstractSchematic illustration of the T7 RNA polymerase-enabled high-density protein display (TIED) on bacterial spores and its unique features as a biocatalyst platform.

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“…The intensity of each fluorophore across the population can be measured concurrently as an approximation of the relative abundance of each species ( Shou, Ram and Vilar, 2007 ; Kusuda, Shimizu and Toya, 2021 ) or by measuring fluorescence of many individual cells e.g., through flow cytometry ( Aditya et al, 2021 ; Konstantinidis et al, 2021 ) or microfluidics systems with microscopes ( Chait et al, 2017 ) (outputs in Figure 2 ). Fluorescence has additional benefits: it provides spatial information in heterogenous environments ( Chen et al, 2015 ; Kozlowski et al, 2021 ; Krishna Kumar et al, 2021 ) and computational tools with integrated image analysis and control algorithms have already been developed ( Pedone et al, 2021 ). However, it also has drawbacks as a method of measuring community composition: fluorescent intensity is an imperfect proxy for population data as there can be variations in expression levels and delays [due to folding kinetics ( Pédelacq et al, 2006 )] between reporters.…”

Section: Control Output: Measuring Community Compositionmentioning

confidence: 99%

Cybergenetic control of microbial community composition

Lee

Steel²

2022

Front. Bioeng. Biotechnol.

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The use of bacterial communities in bioproduction instead of monocultures has potential advantages including increased productivity through division of labour, ability to utilise cheaper substrates, and robustness against perturbations. A key challenge in the application of engineered bacterial communities is the ability to reliably control the composition of the community in terms of its constituent species. This is crucial to prevent faster growing species from outcompeting others with a lower relative fitness, and to ensure that all species are present at an optimal ratio during different steps in a biotechnological process. In contrast to purely biological approaches such as synthetic quorum sensing circuits or paired auxotrophies, cybergenetic control techniques - those in which computers interface with living cells-are emerging as an alternative approach with many advantages. The community composition is measured through methods such as fluorescence intensity or flow cytometry, with measured data fed real-time into a computer. A control action is computed using a variety of possible control algorithms and then applied to the system, with actuation taking the form of chemical (e.g., inducers, nutrients) or physical (e.g., optogenetic, mechanical) inputs. Subsequent changes in composition are then measured and the cycle repeated, maintaining or driving the system to a desired state. This review discusses recent and future developments in methods for implementing cybergenetic control systems, contrasts their capabilities with those of traditional biological methods of population control, and discusses future directions and outstanding challenges for the field.

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Genetically Programmable Microbial Assembly

Cited by 13 publications

References 47 publications

Stress-Tolerant, Recyclable, and Renewable Biocatalyst Platform Enabled by Engineered Bacterial Spores

Stress-Tolerant, Recyclable, and Renewable Biocatalyst Platform Enabled by Engineered Bacterial Spores

Stress-tolerant, recyclable, and autonomously renewable biocatalyst platform enabled by engineered bacterial spores

Cybergenetic control of microbial community composition

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