Part:BBa_K4907121
cspA promoter-cspA 5'-UTR-TEE-sspC-vsw-3 rnapC-cspA 3'-UTR-B0015
Biology
pCspA
pCspA is the promoter of CspA which is a type of cold shock protein. 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).
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
For the final goal of constructing AND-logic gate based on the split VSW-3 RNAP to reduce the leakage of cspA-mRNA expression system, we have endeavored verifying every key point involved in the AND gate. Due to the low-temperature preference of VSW-3 RNAP, it was convinced that the low-temperature (25 °C in this test) would be set as an intrinsic input of the multi-input AND-logic gate. However, we met some challenges when cloning VSW-3 RNAPN-NpuN into the CspA CREC system, so we decided to clone this sequence into the classic cold-inducible vector (8). pCold I, in which the cold-responsive mechanism is also based on the function of cspA-mRNA that just distinguishes from the CspA CREC in few sequences.
Characterization
Agarose gel electrophoresis (AGE)
When building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1201 bp (lane K4907130).
Characterization of three inputs
At first, we tried to co-transform the pCold I-VSW-3 RNAPN-NpuN (BBa_K4907148_pCold I), L-arabinose-induced SspC-VSW-3 RNAPC expression circuit (BBa_K4907116_pSB1C3) and the reporting circuit of the pVSW-3(18) promoter (BBa_K4907121_pSB3K3) into BL21(DE3). It should be noted that the cspA promoter on pCold I is an IPTG-inducible one because a copy of lacO is placed downstream the promoter sequence. Hence, in accordance with the ways of induction, the AND gate constructed here was a three-input one, linking the chemogenetics and thermogenetics (Fig. 3a, left). We set carefully the conditions of different control groups and measured the output signals after induction for 12 hours. As expected, when all the induction requirements were met (0.5 mM IPTG, 0.2% L-arabinose (m/v), cultivated at 25 °C), the normalized fluorescence intensity was significantly strongest (Fig. 3a, right). Besides, when one input was absent (“0”), the output signals were even about 4-fold lower than the “all-input-1” group and all these deficient groups exhibited an equal level of the weak output signals, which indicated that the three-input AND gate was very stringent.
Then, the L-arabinose-induced SspC-VSW-3 RNAPC expression circuit (BBa_K4907116_pSB1C3) was replaced by CspA CREC-SspC-VSW-3 RNAPC (BBa_K4907121_pSB1C3), in which the split half of RNA polymerase is under the control of cspA-mRNA system that needs no inducers at all. Therefore, a two-input AND gate was formed (Fig. 13b, left). Similar experiment was performed as mentioned above while the input of arabinose was removed, and the results demonstrated that the pVSW-3(18) promoter could be activated only when both genes of split halves were induced by IPTG and the low-temperature (Fig. 3b, right). Given the conditions that IPTG was added, only the relative low cultivating temperature (25 °C) would result in the “1” state of output. In other word, the strategy of leveraging the tight control of AND-logic gate to reduce the leakage expression of CspA CREC system was sufficiently feasible. In summary, combining CspA CREC and the split intein VSW-3 RNA polymerase can generate a modular and orthogonal genetic multi-input AND-logic gate like the hrp system reported before (9). What’s more, we believed that by replacing the input promoters and introducing another logic gates into it, the applications of the split intein VSW-3 RNA polymerase will be further developed and expanded.
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).
8.G. Qing et al., Cold-shock induced high-yield protein production in Escherichia coli. Nature Biotechnology 22, 877-882 (2004).
9.B. Wang, R. I. Kitney, N. Joly, M. Buck, Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nature Communications 2, 508 (2011).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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