Difference between revisions of "Part:BBa K4907111"

Revision as of 16:50, 10 October 2023

pVSW-3(16)-B0034-rfp-B0015

Biology

pVSW-3(16)

Some RNA polymerases of eukaryotes and viruses have domains that specifically recognize DNA base sequences, and they are specifically matched with their corresponding promoters (1). VSW-3 RNAP is encoded by the psychrophilic phage VSW-3 in plateau lakes and has low temperature specificity (2). Hengxia et al. characterized pVSW-3 series promoters for the first time and pVSW-3(16) is one of them.

Usage and Design

In order to construct a matching expression system of the VSW-3 RNAP, we characterized its potentially useful promoters using RFP (BBa_K4907037) as the reporter. pVSW-3(16) is one of the more efficient promoters in the series. Different sub parts were assembled into pSB3K3 plasmid backbone to get the composite part BBa_K4907110 (Fig. 1). The plasmid was transformed into E. coli DH5α and the positive transformants were confirmed by kanamycin, colony PCR and sequencing.

Characterization

Agarose gel electrophoresis (AGE)

When building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1220bp (lane K4907110).

Comparison of series promoters from pVSW-3(19) to pVSW-3(16)

The regulatory plasmid containing VSW-3 RNAP and the expressive plasmids with different promoters were transformed into E. coli BL21(DE3). The correct dual-plasmid system was confirmed by chloramphenicol and kanamycin. We characterized the series promoters from pVSW-3(19) to pVSW-3(16) using RFP under 25 ℃. As shown in Fig. 3, pVSW-3(19), pVSW-3(18), and pVSW-3(16) showed better than pVSW-3(16).

Reference

1. S. Borukhov, E. Nudler, RNA polymerase: the vehicle of transcription. Trends in Microbiology 16, 126-134 (2008).

2. H. Xia et al., Psychrophilic phage VSW-3 RNA polymerase reduces both terminal and full-length dsRNA byproducts in in vitro transcription. RNA Biology 19, 1130-1142 (2022).

Sequence and Features