Part:BBa_K4907119

pCspA-cspA 5'-UTR-TEE-VSW-3 rnap-cspA 3'-UTR-B0015

Biology

pCspA

pCspA is the promoter of CspA which is a type of cold shock proteins. When E. coli is transferred to low temperatures, the cells exhibit an adaptive response to the temperature downshift. More specifically, cold shock starts the expression of a set of proteins defined as cold shock proteins which have been shown to play important roles in protein synthesis at low temperatures (1).

cspA 5’-UTR

Between the 5'end and the coding sequence is a short region that is not translated—the 5'-untranslated region or 5'-UTR. As for cspA 5’-UTR, its stability has been shown to play a major role in the cold shock expression of CspA (2). Experiments have shown that the mechanism of cspA cold-responsive element (CRE) is not related to the cspA promoter, while the 5’-UTR plays a greater role in the induction of downstream genes’ expression due to its conformational change (3).

TEE

TEE refers to translation-enhancing element. This sequence is preferentially bound by ribosomes initiating translation. So once bound to the TEE, ribosomes are rarely available to translate other mRNAs (4).

cspA 3’-UTR

Similarly, 3’-UTR is defined as the untranslated region at the 3’end of mRNA. The stability of 3’-UTR has been shown to play a major role in cspA CRE because of the interaction between mRNA 5’-UTR and 3’-UTR.

VSW-3 RNAP

The VSW-3 RNAP is a novel single-subunit RNA polymerase encoded by the chillophilic phage VSW-3, which was first characterized in vitro in 2022. VSW-3 RNAP showed a good low-temperature performance, producing fewer terminal and full-length dsRNA byproducts than the T7 RNAP transcript in vitro (5). Moreover, the in vitro transcription products of VSW-3 RNAP were used to prepare mRNA for mRNA therapy in vivo due to the superior protein expression levels of VSW-3 RNA transcripts, compared to T7 RNAP transcripts (6).

Usage and design

The efficiency of VSW-3 RNAP when loaded into the CspA CREC system (please see more information in BBa_K4907118) should be determined, since we planned to achieve a design with AND-logic gate to reduce pCspA leakage. Therefore, we constructed the pCspA-B0034-VSW-3 RNAP-B0015 circuit (CspA CREC-VSW-3 RNAP) on the backbone pSB1C3 (BBa_K4907119_pSB1C3) and co-transformed this plasmid with the reporting circuit of pVSW-3(18) (BBa_K4907109_pSB3K3) into E. coli BL21(DE3).

Fig. 1 Gene circuit of BBa_K4907119_pSB1C3

Characterization

Agarose gel electrophoresis (AGE)

When building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-3226 bp (lane K4907119).

Fig. 2 The result of colony PCR. Plasmid BBa_K4907119_pSB1C3

Fluorescence characterization at 20℃

After induction at 20 °C for 20 hours, even the temperature might not be the optimal temperature for VSW-3 RNAP, the output signal of the RNA polymerase loaded into CspA CREC was much stronger than that of the control group without harboring VSW-3 RNAP (Fig. 12). Hence, it was convinced that the combination of VSW-3 RNAP with CspA CREC could function well. It was very promising to achieve the goal of constructing a cold-responsive transcriptional AND gate.

Fig. 3 Characterizations for testing the efficiency of VSW-3 RNAP combined with CspA CREC system at 20 °C.

Reference

1. W. Bae, P. G. Jones, M. Inouye, CspA, the major cold shock protein of Escherichia coli, negatively regulates its own gene expression. Journal of Bacteriology 179, 7081-7088 (1997).

2. L. Fang, W. Jiang, W. Bae, M. Inouye, Promoter-independent cold-shock induction of cspA and its derepression at 37°C by mRNA stabilization. Molecular Microbiology 23, 355-364 (1997).

3. A. Hoynes-O'Connor, K. Hinman, L. Kirchner, T. S. Moon, De novo design of heat-repressible RNA thermosensors in E. coli. Nucleic Acids Research 43, 6166-6179 (2015).

4. G. Qing et al., Cold-shock induced high-yield protein production in Escherichia coli. Nature Biotechnology 22, 877-882 (2004).

5. 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).

6. G. Wang et al., mRNA produced by VSW-3 RNAP has high-level translation efficiency with low inflammatory stimulation. Cell Insight 1, 100056 (2022).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 931
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 454