Codon-Restrained Method for Both Eliminating and Creating Intragenic Bacterial Promoters

Logel, Dominic Y.; Trofimova, Ellina; Jaschke, Paul R.

doi:10.1021/acssynbio.1c00359

Cited by 8 publications

(5 citation statements)

References 58 publications

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“…An advantage of this method over other alternative biocontainment methods is that changing the start codon of a gene from ATG to TAG is unlikely to disrupt any intragenic promoters in the affected gene sequence [66,67]. Furthermore, although the removal of entire essential genes from phages has been demonstrated to work efficiently [68], the method requires prior knowledge of the gene function and overall phage biology to ensure viability is not compromised.…”

Section: Discussionmentioning

confidence: 99%

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Designing a simple and efficient phage biocontainment system using the amber suppressor initiator tRNA

Tsoumbris,

Vincent,

Jaschke

2024

Preprint

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Multidrug-resistant infections are becoming increasingly prevalent worldwide. One of the fastest emerging alternative and adjuvant therapies being proposed is phage therapy. Naturally-isolated phages are used in the vast majority of phage therapy treatments today. Engineered phages are being developed to enhance the effectiveness of phage therapy, but concerns over their potential escape remains a salient issue. To address this problem, we designed a biocontained phage system based on conditional replication using amber stop codon suppression. This system can be easily installed on any natural phage with a known genome sequence. To test the system, we mutated the start codons of three essential capsid genes in the phage φX174 to the amber stop codon (TAG). These phages were able to efficiently infect host cells expressing the amber initiator tRNA, which suppresses the amber stop codon and initiates translation at TAG stop codons. The amber phage mutants were also able to successfully infect host cells and reduce their population on solid agar and liquid culture but could not produce infectious particles in the absence of the amber initiator tRNA or complementing capsid gene. We did not detect any growth-inhibiting effects on E. coli strains known to lack a receptor for φX174, and we show that engineered phages have a limited propensity for reversion. The approach outlined here may be useful to control engineered phage replication in both the lab and clinic.

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Section: Discussionmentioning

confidence: 99%

“…Furthermore, the removal of genes may alter viability as some phages are extremely sensitive to genome shortening [73]. Additionally, there is the potential for disruption to essential intragenic regulatory sequences, impacting adjacent gene expression [67,74].…”

Section: Discussionmentioning

confidence: 99%

Designing a simple and efficient phage biocontainment system using the amber suppressor initiator tRNA

Tsoumbris,

Vincent,

Jaschke

2024

Preprint

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“…reduce changes in promoter activity, regulatory functions, and translation rate and avoid generating unwanted cryptic promoters and translated ORFs. The development of predictive models capable of forecasting transcriptional and translational effects and integrating these predictions to refactor codon composition is expected to minimize the negative fitness impact of synonymous genome recoding [83][84][85][86][87][88][89][90][91][92] .…”

Section: Future Genome Design Projects Should Focus On Developing And...mentioning

confidence: 99%

Synthetic genomes unveil the effects of synonymous recoding

Nyerges,

Chiappino-Pepe,

Budnik

et al. 2024

Preprint

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Engineering the genetic code of an organism provides the basis for (i) making any organism safely resistant to natural viruses and (ii) preventing genetic information flow into and out of genetically modified organisms while (iii) allowing the biosynthesis of genetically encoded unnatural polymers1–4. Achieving these three goals requires the reassignment of multiple of the 64 codons nature uses to encode proteins. However, synonymous codon replacement—recoding—is frequently lethal, and how recoding impacts fitness remains poorly explored. Here, we explore these effects using whole-genome synthesis, multiplexed directed evolution, and genome-transcriptome-translatome-proteome co-profiling on multiple recoded genomes. Using this information, we assemble a syntheticEscherichia coligenome in seven sections using only 57 codons to encode proteins. By discovering the rules responsible for the lethality of synonymous recoding and developing a data-driven multi-omics-based genome construction workflow that troubleshoots synthetic genomes, we overcome the lethal effects of 62,007 synonymous codon swaps and 11,108 additional genomic edits. We show that synonymous recoding induces transcriptional noise including new antisense RNAs, leading to drastic transcriptome and proteome perturbation. As the elimination of select codons from an organism’s genetic code results in the widespread appearance of cryptic promoters, we show that synonymous codon choice may naturally evolve to minimize transcriptional noise. Our work provides the first genome-scale description of how synonymous codon changes influence organismal fitness and paves the way for the construction of functional genomes that provide genetic firewalls from natural ecosystems and safely produce biopolymers, drugs, and enzymes with an expanded chemistry.

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“…The reason for this selection was its strict σ S selectivity [43,45]. It is well known that the difference in the −35 hexamer, spacer region, and discriminator can result in different σ S selectivity and promoters' strength [18,40,[46][47][48][49][50][51]. Therefore, we carried out saturation mutations in the above regions to obtain a continuous intensity spectrum of the response σ S factor.…”

Section: Introductionmentioning

confidence: 99%

Enhancing the Activity of a Self-Inducible Promoter in Escherichia coli through Saturation Mutation and High-Throughput Screening

Tong

Amin

et al. 2023

Fermentation

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The use of self-inducible promoters is a promising strategy to address metabolic imbalances caused by overexpression. However, the low activity of natural self-inducible promoters hinders their widespread application. To overcome this limitation, we selected the fic promoter as a model promoter to create an enhanced self-inducible promoter library using saturation mutations and high-throughput screening. Sequence analysis revealed that these promoters share certain characteristics, including semi-conservation in the −35 hexamer, highly conserved cytosine in the −17 motif (compared to −13 for other promoters), and moderate A+T content between positions −33 and −18 in the spacer region. Additionally, the discriminator region of these promotors features high A+T content in the first five bases. We identified PficI-17, PficII-33, and PficIII-14 promoters as the optional promoters in the −35 hexamer, spacer region, and discriminator mutation libraries, respectively. These promotors were used as representatives to measure the specific fluorescence and OD600 nm dynamics in different media and to confirm their effect on the expression of different proteins, including egfp (enhanced green fluorescence protein) and rfp (red fluorescence protein). Overall, our findings provide valuable guidance for modifying promoters and developing a promoter library suitable for regulating target genes.

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Codon-Restrained Method for Both Eliminating and Creating Intragenic Bacterial Promoters

Cited by 8 publications

References 58 publications

Designing a simple and efficient phage biocontainment system using the amber suppressor initiator tRNA

Designing a simple and efficient phage biocontainment system using the amber suppressor initiator tRNA

Synthetic genomes unveil the effects of synonymous recoding

Enhancing the Activity of a Self-Inducible Promoter in Escherichia coli through Saturation Mutation and High-Throughput Screening

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