Part:BBa_K4907120

pCspA-cspA 5'-UTR-TEE-VSW-3 rnapN-npuN-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 temperature, 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 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).

VSW-3 RNAPN-NpuN and SspC VSW-3 RNAPC

Based on the split-intein (7) combined with the novel VSW-3 system. In our design, the VSW-3 RNAP was split into two halves and fused to the split intein SspC and NpuN respectively.

Usage and design

We hope to construct this circuit to further characterize AND gate. But unfortunately, due to time constraints, we chose other better schemes and shelved this circuit.

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).

7.L. Saleh, F. B. Perler, Protein splicing in cis and in trans. Chem Rec 6, 183-193 (2006).

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 1692
  Illegal BglII site found at 1929
  
• 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
  Illegal BsaI.rc site found at 1685