A set of temperature sensitive-replication/-segregation and temperature resistant plasmid vectors with different copy numbers and in an isogenic background (chloramphenicol, kanamycin, lacZ, repA, par, polA)

Hashimoto-Gotoh, Tamotsu; Yamaguchi, Miki; Yasojima, Koji; Tsujimura, Akira; Wakabayashi, Yoshihiro; Watanabe, Yoshihisa

doi:10.1016/s0378-1119(99)00434-5

Cited by 74 publications

(36 citation statements)

References 31 publications

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“…in E. coli), it failed to work efficiently in V. parahaemolyticus. Therefore, plasmid pLM2274 was subsequently linearized with SphI and cloned into a gentamicin-resistant derivative of plasmid pKF38ts1 (Hashimoto-Gotoh et al, 2000). Plasmid pKF38ts1 contains a temperature-sensitive pSC101 origin of replication, which allowed introduction and maintenance of the transposon-bearing plasmid Tn5cat-TS in V. parahaemolyticus TR strain LM5674 at 30∞C.…”

Section: Transposon Mutagenesismentioning

confidence: 99%

Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus

Enos-Berlage

Güvener

Keenan

et al. 2005

Molecular Microbiology

206

193

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SummaryVibrio parahaemolyticus isolates display variation in colony morphology, alternating between opaque (OP) and translucent (TR) cell types. Phase variation is the consequence of genetic alterations in the locus encoding the quorum sensing output regulator OpaR. Here, we show that both cell types form stable, but distinguishable biofilms that differ with respect to attachment and detachment profiles to polystyrene, pellicle formation and stability at the air/medium interface, and submerged biofilm architecture and dispersion at a solid/liquid interface. The pellicle, which is a cohesive mat of cells, was exploited to identify mutants having altered or defective biofilm formation. Transposon insertion mutants were obtained with defects in genes affecting multiple cell surface characteristics, including extracellular polysaccharide, mannose-sensitive haemagglutinin type 4 pili and polar (but not lateral) flagella. Other insertions disrupted genes coding for potential secreted proteins or transporters of secreted proteins, specifically haemolysin co-regulated protein and an RTX toxin-like membrane fusion transporter, as well as potential modifiers of cell surface molecules ( nagAC operon). The pellicle screen also identified mutants with lesions in regulatory genes encoding H-NS, a CsgD-like repressor and an AraC-like protein. This work initiates the characterization of V. parahaemolyticus biofilm formation in the OP and TR cell types and identifies a diverse repertoire of cell surface elements that participate in determining multicellular architecture.

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Section: Transposon Mutagenesismentioning

confidence: 99%

Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus

Enos-Berlage

Güvener

Keenan

et al. 2005

Molecular Microbiology

206

193

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“…Next, a gentamicin-resistant cassette (GM) isolated from pBSL142 17) by digestion with HindIII was inserted into the HindIII site within the avtA gene of pAvtA18. The PvuII fragment of the resulting plasmid containing the disrupted avtA gene was purified and cloned into the SmaI site of pTH18ks1 18) to yield pAvtA18ks-GM. Transformants harboring pAvtA18ks-GM were grown in L-broth at 42 C overnight, and integrants were selected on L-agar plates containing 6.25 mg/mL of gentamicin.…”

Section: Methodsmentioning

confidence: 99%

“…Next, a kanamycin-resistant cassette (KM) isolated from pBSL97 17) was inserted into the SacII site in the yfbQ gene, followed by subcloning of the BamHI fragment with the disrupted yfbQ gene in pTH18cs1 18) to yield pYfbQ18cs-KM. This plasmid was used to transform E. coli HYE011 (avtA::GM) in order to construct a double knockout mutant HYE021 (avtA::GM, yfbQ::KM), as described above.…”

Section: Methodsmentioning

confidence: 99%

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Isolation of a Mutant Auxotrophic forL-Alanine and Identification of Three Major Aminotransferases That SynthesizeL-Alanine inEscherichia coli

Yoneyama

Hori

Lim

et al. 2011

Bioscience, Biotechnology, and Biochemistry

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“…The construct was then made temperature sensitive by mutating the residue Ala56(gct) to Val56(gtt) in the repA gene of pSC101 through sitedirected mutagenesis using Stratagene's QuikChange site-directed mutagenesis protocol. 19 pSEL1 and pSEL2 plasmids containing the I-CreI target site (5 0 CAA AAC GTC GTG AGA CAG TTT GGT 3 0 ) were created as follows. 16 The I-CreI insert was prepared by annealing the phosphorylated primers (5 0 Phos AGC TCA AAA CGT CGT GAG ACA GTT TGG T 3 0 ) and (5 0 Phos AGC TAC CAA ACT GTC TCA CGA CGT TTT G 3 0 ) and then inserting this fragment into the HindIII site of the pSEL1 and pSEL2 plasmids.…”

Section: Cloning Of Controls Used In Cellular Fractionationmentioning

confidence: 99%

Genetic selection system for improving recombinant membrane protein expression in E. coli

et al. 2009

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A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C-terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I-CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75-fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90-fold.

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A set of temperature sensitive-replication/-segregation and temperature resistant plasmid vectors with different copy numbers and in an isogenic background (chloramphenicol, kanamycin, lacZ, repA, par, polA)

Cited by 74 publications

References 31 publications

Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus

Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus

Isolation of a Mutant Auxotrophic forL-Alanine and Identification of Three Major Aminotransferases That SynthesizeL-Alanine inEscherichia coli

Genetic selection system for improving recombinant membrane protein expression in E. coli

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