Experimental tools to reduce the burden of bacterial synthetic biology

Grob, Alice; Blasi, Roberto Di; Ceroni, Francesca

doi:10.1016/j.coisb.2021.100393

Cited by 11 publications

(12 citation statements)

References 74 publications

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Section: Discussionmentioning

confidence: 99%

“…We were surprised that this did not lead to extensive mutations to the genome and to the payload device. We have observed in other systems that high levels of recombinant gene expression lead to rapid evolutionary breaking of the genetic device and secondary mutations to the genome to mitigate its impact [35,65,83,84], likely due to its draw on resources, particularly ribosomes [85]. It is likely that the long-term persistence of B. frigoritolerans is enabled through spore formation, initiated in the drier environment, and then allowing spore to be maintained in the soil for long times and then re-accessed by germination when rehydrated [86].…”

Section: Plos Onementioning

confidence: 99%

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Parallel engineering of environmental bacteria and performance over years under jungle-simulated conditions

et al. 2022

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Engineered bacteria could perform many functions in the environment, for example, to remediate pollutants, deliver nutrients to crops or act as in-field biosensors. Model organisms can be unreliable in the field, but selecting an isolate from the thousands that naturally live there and genetically manipulating them to carry the desired function is a slow and uninformed process. Here, we demonstrate the parallel engineering of isolates from environmental samples by using the broad-host-range XPORT conjugation system (Bacillus subtilis mini-ICEBs1) to transfer a genetic payload to many isolates in parallel. Bacillus and Lysinibacillus species were obtained from seven soil and water samples from different locations in Israel. XPORT successfully transferred a genetic function (reporter expression) into 25 of these isolates. They were then screened to identify the best-performing chassis based on the expression level, doubling time, functional stability in soil, and environmentally-relevant traits of its closest annotated reference species, such as the ability to sporulate and temperature tolerance. From this library, we selected Bacillus frigoritolerans A3E1, re-introduced it to soil, and measured function and genetic stability in a contained environment that replicates jungle conditions. After 21 months of storage, the engineered bacteria were viable, could perform their function, and did not accumulate disruptive mutations.

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Section: Discussionmentioning

confidence: 99%

Section: Plos Onementioning

confidence: 99%

Parallel engineering of environmental bacteria and performance over years under jungle-simulated conditions

et al. 2022

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“…To engineer more robust components, directed evolution experiments should be designed well outside of 'burden territory', in this way avoiding the effects of multi-layered burden-related retroactivity on induction dynamics. Designing for lower metabolic burden will prevent the evolution of such context-dependent properties (Figure 5b) and can be undergone in various ways [64], for example, using software tools to predict transcriptional or translational efficiencies of synthetic constructs [21,24,44,45,65], or coupling circuit expression with genetically encoded sensors of metabolic burden as feedback controllers [25,64]. Incorporating such steps into construct design will ensure that the complexity of robust synthetic circuits continues to grow.…”

Section: Discussionmentioning

confidence: 99%

Reducing metabolic burden in the PACEmid evolver system by remastering high‐copy phagemid vectors

Davenport

Tica

Isalan

2022

Engineering Biology

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Orthogonal or non‐cross‐reacting transcription factors are used in synthetic biology as components of genetic circuits. Brödel et al. (2016) engineered 12 such cIλ transcription factor variants using a directed evolution ‘PACEmid’ system. The variants operate as dual activator/repressors and expand gene circuit construction possibilities. However, the high‐copy phagemid vectors carrying the cIλ variants imposed high metabolic burden upon cells. Here, the authors ‘remaster’ the phagemid backbones to relieve their burden substantially, exhibited by a recovery in Escherichia coli growth. The remastered phagemids' ability to function within the PACEmid evolver system is maintained, as is the cIλ transcription factors' activity within these vectors. The low‐burden phagemid versions are more suitable for use in PACEmid experiments and synthetic gene circuits; the authors have, therefore, replaced the original high‐burden phagemids on the Addgene repository. The authors’ work emphasises the importance of understanding metabolic burden and incorporating it into design steps in future synthetic biology ventures.

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“…Downstream, this can negatively impact the efficiency of delivery and therefore, efficacy. However, some encouraging studies explore methods to circumnavigate the fitness burden imposed by synthetic gene circuits, which are often used to control the expression of therapeutics [ 9 ]. For example, orthogonal ribosomes engineered with synthetic 16S rRNA can only translate genes within the synthetic circuit, while host ribosomes can translate both host genes and circuit genes [ 9 , 10 ].…”

Section: Secretionmentioning

confidence: 99%