Part:BBa_K4907108

pVSW-3 (18)-B0034-rfp-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 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(18) 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(18) 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.

Fig. 1 Gene circuit of pVSW-3 series promoter reporting circuit

Characterization

Agarose gel electrophoresis (AGE)

When building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1199 bp (lane K4907108).

Fig. 2 The result of colony PCR. Plasmid BBa_K4907108_pSB3K3

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(17) showed better than pVSW-3(16).

Fig. 3 The comparison of normalized fluorescence intensity the series promoters from pVSW-3(19) to pVSW-3(16).

The optimal temperature of VSW-3 system

Since the low-temperature inducible expression, or a cold-responsive expression pattern, was the need for achieving the goal of anti-icing, the optimal temperature of the VSW-3 system should be investigated as well. The bacteria (BL21(DE3)) harboring VSW-3 RNAP and the reporting circuits of pVSW-3(18) was cultivated at different temperatures after induction respectively. Among the temperatures we tested, the VSW-3 system has the strongest activity at 25 °C (at least 4-fold higher than other groups) (Fig. 4), which was consistent with the results in vitro (3). Besides, the VSW-3 RNAP functioned better at 15 °C than 30 °C and physiological 37 °C, which implied the obvious low-temperature preference of this RNA polymerase. Due to the limited time and the number of available thermostats in our lab, more detailed temperature gradients need to be examined and we hoped that this could be achieved in the future.

Fig. 4 Characterizations of the temperature effect for VSW-3 system (a) and T7 system (b).

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).

3.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
  
• 1000
  COMPATIBLE WITH RFC[1000]