Positive and negative selection using the <i>tetA-sacB</i> cassette: recombineering and P1 transduction in <i>Escherichia coli</i>

Li, Xintian; Thomason, Lynn C.; Sawitzke, James A.; Costantino, Nina; Court, Donald L.

doi:10.1093/nar/gkt1075

Cited by 162 publications

(173 citation statements)

References 32 publications

(27 reference statements)

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“…3 and Supplementary Information. The Cas9-binding RNA structure and transcription terminator of the sgRNA are already encoded within the chromosome, and are linked to a counter-selectable marker, tet - sacB 28. Once the homology targeting sgRNA sequence is inserted by replacing tet - sacB , down-regulation of the targeted gene is accomplished by adding arabinose to the culture medium.…”

Section: Resultsmentioning

confidence: 99%

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tCRISPRi: tunable and reversible, one-step control of gene expression

Jun

Erickstad

et al. 2016

Sci Rep

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The ability to control the level of gene expression is a major quest in biology. A widely used approach employs deletion of a nonessential gene of interest (knockout), or multi-step recombineering to move a gene of interest under a repressible promoter (knockdown). However, these genetic methods are laborious, and limited for quantitative study. Here, we report a tunable CRISPR-cas system, “tCRISPRi”, for precise and continuous titration of gene expression by more than 30-fold. Our tCRISPRi system employs various previous advancements into a single strain: (1) We constructed a new strain containing a tunable arabinose operon promoter PBAD to quantitatively control the expression of CRISPR-(d)Cas protein over two orders of magnitude in a plasmid-free system. (2) tCRISPRi is reversible, and gene expression is repressed under knockdown conditions. (3) tCRISPRi shows significantly less than 10% leaky expression. (4) Most important from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step of oligo recombineering. Our results show that tCRISPRi, in combination with recombineering, provides a simple and easy-to-implement tool for gene expression control, and is ideally suited for construction of both individual strains and high-throughput tunable knockdown libraries.

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Section: Resultsmentioning

confidence: 99%

“…The lambda Red recombineering was performed using lambda recombination functions provide by pSIM18 following published methods4849, tet - sacB selection and counter-selection was described previously28, P1 transduction was carried out according to published methods50.…”

Section: Methodsmentioning

confidence: 99%

tCRISPRi: tunable and reversible, one-step control of gene expression

Jun

Erickstad

et al. 2016

Sci Rep

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“…The positive selection was performed using the rich medium supplemented with tetracycline after the first crossover recombination. Then, the second crossover recombination was performed, and a mutant with the gene deleted was selected through the counter‐selection using the rich medium supplemented with sucrose (X. Li, Thomason, Sawitzke, Costantino, & Court, ), which was confirmed by PCR. For gene overexpression/complementation, promoters and target genes were amplified separately by PCR.…”

Section: Methodsmentioning

confidence: 99%

Contribution of cellulose synthesis, formation of fibrils and their entanglement to the self‐flocculation of Zymomonas mobilis

Xia

Liu

Zhao

et al. 2018

Biotech & Bioengineering

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Due to the unique Entner-Doudoroff pathway, Zymomonas mobilis has been acknowledged as a potential host to be engineered for biorefinery to produce biofuels and biobased chemicals. The self-flocculation of Z. mobilis can make the bacterial cells self-immobilized within bioreactors for high density to improve product productivities, and in the meantime enhance their tolerance to stresses, particularly product inhibition and the toxicity of byproducts released during the pretreatment of lignocellulosic biomass. In this work, we explored mechanism underlying such a phenotype with the self-flocculating strain ZM401 developed from the regular non-flocculating strain ZM4. Cellulase de-flocculation and the restoration of the self-flocculating phenotype for the de-flocculated bacterial cells subjected to culture confirmed the essential role of cellulose biosynthesis in the self-flocculation of ZM401. Furthermore, the deactivation of both Type I and Type IV restriction-modification systems was performed for ZM4 and ZM401 to improve their transformation efficiencies. Comparative genome analysis detected the deletion of a thymine from ZMO1082 in ZM401, leading to a frame-shift mutation for the putative gene to be integrated into the neighboring downstream gene ZMO1083 encoding the catalytic subunit A of cellulose synthase, and consequently created a new gene to encode a larger transmembrane protein BcsA_401 for more efficient synthesis of cellulose as well as the development of cellulose fibrils and their entanglement for the self-flocculation of the mutant. These speculations were confirmed by the morphological observation of the bacterial cells under scanning electron microscopy, the impact of the gene deletion on the self-flocculation of ZM401, and the restoration of the self-flocculating phenotype of ZM401 ΔbcsA by the gene complementation. The progress will lay a foundation not only for fundamental research in deciphering molecular mechanisms underlying the self-flocculation of Z. mobilis and stress tolerance associated with the morphological change but also for technological innovations in engineering non-flocculating Z. mobilis and other bacterial species with the self-flocculating phenotype.

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“…Alternatively, the strain XTL298 contains the highly selective tetA-sacB counterselection cassette integrated into the chromosome at the araD locus (227). Plasmids (or strains) containing these cassettes can be used as templates for PCR to generate substrates for recombineering.…”

Section: Precise Deletionsmentioning

confidence: 99%

λ Recombination and Recombineering

Murphy

2016

EcoSal Plus

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The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics.

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Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli

Cited by 162 publications

References 32 publications

tCRISPRi: tunable and reversible, one-step control of gene expression

tCRISPRi: tunable and reversible, one-step control of gene expression

Contribution of cellulose synthesis, formation of fibrils and their entanglement to the self‐flocculation of Zymomonas mobilis

λ Recombination and Recombineering

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