CJW5461 strain菌株 BioVector NTCC质粒载体菌种细胞基因保藏中心

BioVector® NTCC® CJW5461 strain菌株

CJW5461 MG1655 ∆lacYA A clean deletion of lacYA on the chromosome was done by two-step recombination (Datsenko and Wanner, 2000) with cat-sacB to leave no scar (Sawitzke et al., 2013). As a result, the chromosomal region after the lacZ stop codon to the lacA stop codon was deleted, and the lacZ stop codon is followed by the 3’ untranslated region of lacA. CJW5616 MG1655 ∆lacIZYA/pEXT22- lacI-lacZYA The chromosomal region spanning lacI and lacZYA was amplified from MG1655 and introduced into pEXT22, a low copy plasmid (Dykxhoorn et al., 1996). Plasmid was then electroporated into CJW6643. This strain is resistant to kanamycin. CJW6643 MG1655 ∆lacIZYA The chromosomal region of lacI and lacZYA was deleted by insertion of a kanamycin resistance gene (amplified from pKD13) via lambda Red recombination. The cassette was then removed by FLP recombinase as previously described (Datsenko and Wanner, 2000). This strain was used as a background strain when lac operon was needed to be present only on the plasmid. CJW6647 MG1655 ∆lacIZYA/pUC19- lacI-lacZ The chromosomal region spanning lacI and lacZ was amplified from CJW5461 and introduced into pUC19 via Gibson assembly (Gibson et al., 2009). Plasmid was then electroporated into CJW6643. This strain was used for the “No gfp” control in Figure 6. This strain is resistant to ampicillin. CJW6770 DH5α/pUC19-lacIPlacUV5-lacZYA The chromosomal region spanning lacI and lacZYA was introduced into the pUC19 backbone by multifragment Gibson assembly (Gibson et al., 2009). In preparation of the fragment containing lacZYA, Plac was changed into PlacUV5 by using a primer with twobase mismatches. As a result, TATGT in the -10 region was changed to TATAA. The plasmid was used for in vitro transcription of lacZ with purified E. coli RNAP holoenzyme. This strain is resistant to ampicillin. CJW6919 MG1655/pUC19- Ptet-gfp Inspired by the Ptet-based overexpression system made previously (Hui et al., 2015), tetR and gfp genes are driven by Ptet. They are inserted into pUC19 via Gibson assembly (Gibson et al., 2009). The plasmid was electroporated into MG1655. This strain is resistant to ampicillin. 2 CJW6920 MG1655/pUC19- Ptet-topA The plasmid was made the same way as described in CJW6919. topA gene was amplified from MG1655 chromosome. This strain is resistant to ampicillin. CJW6986 MG1655/pUC19- Ptet-trpC The plasmid was made the same way as described in CJW6919. trpC gene was amplified from MG1655 chromosome. This strain is resistant to ampicillin. CJW6987 (L = 400) MG1655 ∆lacIZYA/pUC19- lacI(O3-)-LPompA,M1- mgfp(reverse)-lacZ The plasmid was constructed via Gibson assembly (Gibson et al., 2009) of five DNA fragments: pUC19 plasmid backbone, lacI from the chromosome of MG1655, super-folder mgfp with a strong PompA promoter variant (see Figure S7), three transcription terminators—T7t, λ tR2, and rrnB T1—from IR5235 (Irnov and Winkler, 2010), and lacZ from CJW5461. lacO3 overlapping with the stop codon of lacI was intentionally removed during PCR amplification of the lacI fragment. However, lacO3 was included upstream of lacZ in the last DNA fragment (see Figure 6A). The plasmid was then electroporated into CJW6643. This strain was used to test the effect of an upstream transcription unit (gfp) on the transcription of lacZ. mgfp was transcribed in the opposite direction of lacI and lacZ. This strain is resistant to ampicillin. CJW6998 (L = 400) MG1655 ∆lacIZYA/pUC19- lacI(O3-)-LPompA,M5- mgfp(reverse)-lacZ The plasmid was constructed in the same manner as for CJW6987, but with a weak PompA variant (see Figure S7). This strain is resistant to ampicillin. CJW6988 (L = 220) CJW6989 (L = 900) CJW6990 (L = 1400) CJW6991 (L = 1900) CJW6992 (L = 2400) MG1655 ∆lacIZYA/pUC19- lacI(O3-)-LPompA,M1-mgfp (reverse)-lacZ The plasmids were constructed via Gibson assembly (Gibson et al., 2009) of three DNA fragments: (i) a segment of the plasmid in CJW6987 spanning pUC19 plasmid backbone, lacI and gfp, (ii) a linker of variable length (L) amplified from the middle of the topA gene on the chromosome of MG1655 and (iii) lacZ. Each plasmid was then electroporated into CJW6643. These strains are resistant to ampicillin. CJW6999 (L = 220) CJW6993 (L = 900) CJW6994 (L = 1400) CJW6995 (L = 1900) MG1655 ∆lacIZYA/pUC19- lacI(O3-)-LPompA,M5- mgfp(reverse)-lacZ The plasmids were constructed in the same manner as for CJW6988, except that the plasmid in CJW6998 was used to make the first DNA segment for Gibson assembly. This strain is resistant to ampicillin. 3 CJW6996 (L = 2400) MG1655 F-lambda- ilvGrfb-50 rph-1 Used as the wild-type strain.

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