Composite

Part:BBa_K4907113

Designed by: Yannan You   Group: iGEM23_XMU-China   (2023-09-15)
Revision as of 09:14, 11 October 2023 by Haoxihuan (Talk | contribs)


pVSW-3(18)-B0034-rfp-B0034-VSW-3 rnap-B0015

Biology

pVSW-3(18)

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 chillophilic 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(18) is one of them.

VSW-3 RNAP

VSW-3 RNAP is a novel single-subunit RNA polymerase encoded by the chillophilic phage VSW-3 from a plateau lakes. The phage's genome was sequenced in 2017. Heng Xia et al. first characterized VSW-3 RNAP in vitro in 2022. In the work of Heng Xia et al., the VSW-3 RNAP showed good low temperature performance, producing fewer terminal and full-length dsRNA byproducts than the T7 RNAP transcript. Further, Wang Guoquan et al. used VSW-3 RNAP for in vitro transcription to prepare mRNA for mRNA therapy, depending on VSW-3 RNA transcripts exhibiting superior protein expression levels compared to T7 RNAP transcripts(2).

Usage and design

We constructed BBa_K4907113 on pSB3K3. In this circuit, polycistrons formed by red fluorescent protein and VSW-3 RNAP are induced by pVSW-3(18). We hope that VSW-3 RNAP can accumulate continuously with the continuous expression of this circuit, so as to improve the expression efficiency with positive feedback.

Characterization

Agarose gel electrophoresis (AGE)

When we were building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-2761bp (lane K4907113).

Characterization of feedback circuit

The results are shown in Fig. 3. The direct results indicate that the feedback circuit did not work as envisaged, and the experimental bacteria was less fluorescent than the positive control group. We believe that this may be related to the longer length of polycistrons and the greater transcriptional pressure of VSW-3 RNAP. Perhaps placing VSW-3 RNAP downstream to form a single transcription unit induced by pVSW-3(18) to form a new positive feedback circuit could solve this problem, but due to time conbacteriats, we are unable to complete this work for the time being.

Fig. 3 Induction intensity of feedback circuit at 25 ℃

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 474
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 596
    Illegal AgeI site found at 1462
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 985


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