Rapid and Efficient One-Step Metabolic Pathway Integration in <i>E. coli</i>

Bassalo, Marcelo C.; Garst, Andrew D.; Halweg-Edwards, Andrea L.; Grau, William C; Domaille, Dylan W.; Mutalik, Vivek K.; Arkin, Adam P.; Gill, Ryan T.

doi:10.1021/acssynbio.5b00187

Cited by 159 publications

(120 citation statements)

References 35 publications

Supporting

Mentioning

119

Contrasting

Order By: Relevance

“…Genome engineering could be used to protect these pathways from MazF activity56. Optimal regulatory strategies should be designed to balance enhancement of resource redistribution activity and degradation of cellular support subsystems over long time scales.…”

Section: Discussionmentioning

confidence: 99%

Programming mRNA decay to modulate synthetic circuit resource allocation

et al. 2017

View full text Add to dashboard Cite

Synthetic circuits embedded in host cells compete with cellular processes for limited intracellular resources. Here we show how funnelling of cellular resources, after global transcriptome degradation by the sequence-dependent endoribonuclease MazF, to a synthetic circuit can increase production. Target genes are protected from MazF activity by recoding the gene sequence to eliminate recognition sites, while preserving the amino acid sequence. The expression of a protected fluorescent reporter and flux of a high-value metabolite are significantly enhanced using this genome-scale control strategy. Proteomics measurements discover a host factor in need of protection to improve resource redistribution activity. A computational model demonstrates that the MazF mRNA-decay feedback loop enables proportional control of MazF in an optimal operating regime. Transcriptional profiling of MazF-induced cells elucidates the dynamic shifts in transcript abundance and discovers regulatory design elements. Altogether, our results suggest that manipulation of cellular resource allocation is a key control parameter for synthetic circuit design.

show abstract

Section: Discussionmentioning

confidence: 99%

Programming mRNA decay to modulate synthetic circuit resource allocation

et al. 2017

View full text Add to dashboard Cite

show abstract

“…Another method involves using λ-Red recombinase to introduce a "landing pad," which contains I-SceI recognition sites and short sequences homologous to the ends of the desired integration cassette to form a flanking region (FR), into the genome. Subsequently, the cassette containing the genes of interest from a modified donor plasmid is introduced into the FR sites in Scarless (Anders et al, 2014;Bassalo et al, 2016;Chung et al, 2017;Deltcheva et al, 2011;Jiang et al, 2013;Jiang et al, 2015;Li et al, 2015) Note. attB: bacterial phage attachment; CIChE: chemically inducible chromosomal evolution; CIGMC: chromosomal integration of gene(s) with multiple copies; CRIM: conditional-replication, integration, and modular; CRISPR/Cas: clustered regularly interspaced short palindromic repeats/CRISPR-associated system; DSBs: double-strand breaks; FLP: flippase; FR: flanking region; FRT: flippase recombinase target;…”

Section: Molecular Technologies For Integrating Foreign Dna Fragmenmentioning

confidence: 99%

Techniques for chromosomal integration and expression optimization in Escherichia coli

Garcia

Wang

et al. 2018

Biotech & Bioengineering

View full text Add to dashboard Cite

Due to the inherent expression stability and low metabolic burden to the host cell, the expression of heterologous proteins in the bacterial chromosome in a precise and efficient manner is highly desirable for metabolic engineering and live bacterial applications. However, obtaining suitable chromosome expression levels is particularly challenging. In this minireview, we briefly present the technologies available for the integration of heterologous genes into Escherichia coli chromosomes and strategies to optimize the expression levels of heterologous proteins.

show abstract

“…To support their conclusion, the authors also claimed that using the same protocol they successfully inactivated five additional genes. Another recent study describes a CRISPR-based strategy that allowed the integration of entire metabolic pathways in seven distinct loci using ds-DNAs encoding homologous regions to the insertion site of different size with 75–100% efficiencies [19]. …”

Section: Introductionmentioning

confidence: 99%

Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

et al. 2017

View full text Add to dashboard Cite

BackgroundThe exploitation of the CRISPR/Cas9 machinery coupled to lambda (λ) recombinase-mediated homologous recombination (recombineering) is becoming the method of choice for genome editing in E. coli. First proposed by Jiang and co-workers, the strategy has been subsequently fine-tuned by several authors who demonstrated, by using few selected loci, that the efficiency of mutagenesis (number of mutant colonies over total number of colonies analyzed) can be extremely high (up to 100%). However, from published data it is difficult to appreciate the robustness of the technology, defined as the number of successfully mutated loci over the total number of targeted loci. This information is particularly relevant in high-throughput genome editing, where repetition of experiments to rescue missing mutants would be impractical. This work describes a “brute force” validation activity, which culminated in the definition of a robust, simple and rapid protocol for single or multiple gene deletions.ResultsWe first set up our own version of the CRISPR/Cas9 protocol and then we evaluated the mutagenesis efficiency by changing different parameters including sequence of guide RNAs, length and concentration of donor DNAs, and use of single stranded and double stranded donor DNAs. We then validated the optimized conditions targeting 78 “dispensable” genes. This work led to the definition of a protocol, featuring the use of double stranded synthetic donor DNAs, which guarantees mutagenesis efficiencies consistently higher than 10% and a robustness of 100%. The procedure can be applied also for simultaneous gene deletions.ConclusionsThis work defines for the first time the robustness of a CRISPR/Cas9-based protocol based on a large sample size. Since the technical solutions here proposed can be applied to other similar procedures, the data could be of general interest for the scientific community working on bacterial genome editing and, in particular, for those involved in synthetic biology projects requiring high throughput procedures.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0681-1) contains supplementary material, which is available to authorized users.

show abstract

Rapid and Efficient One-Step Metabolic Pathway Integration in E. coli

Cited by 159 publications

References 35 publications

Programming mRNA decay to modulate synthetic circuit resource allocation

Programming mRNA decay to modulate synthetic circuit resource allocation

Techniques for chromosomal integration and expression optimization in Escherichia coli

Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

Contact Info

Product

Resources

About