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| | ===Biology=== | | ===Biology=== |
| − | ====pCspA==== | + | ====pVSW-3(17)==== |
| − | pCspA is the promoter of CspA which is a type of cold shock proteins. When <i>E. coli</i> 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).
| + | 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 <i>et al</i>. characterized pVSW-3 series promoters for the first time and pVSW-3(17) is one of them. |
| − | ====<i>cspA</i> 5′-UTR====
| + | ===Usage and Design=== |
| − | 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 <i>cspA</i> 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 <i>cspA</i> <b>cold-responsive element (CRE)</b> is not related to the <i>cspA</i> promoter, while the 5′-UTR plays a greater role in the induction of downstream genes′ expression due to its conformational change (3).
| + | In order to construct a matching expression system of the VSW-3 RNAP, we characterized its potentially useful promoters using RFP (<partinfo>BBa_K4907037 </partinfo>) as the reporter. pVSW-3(17) is one of the more efficient promoters in the series. Different sub parts were assembled into pSB3K3 plasmid backbone to get the composite part <partinfo>BBa_K4907110</partinfo> (Fig. 1). The plasmid was transformed into <i>E. coli</i> DH5α and the positive transformants were confirmed by kanamycin, colony PCR and sequencing. |
| − | ====TEE==== | + | <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/biaozhen/pvsw-3-all-rfp.png" width="400px"></html></center> |
| − | <b>TEE</b> refers to <b>t</b>ranslation <b>e</b>nhancing <b>e</b>lement. This sequence is preferentially bound by ribosomes initiating translation. So once bound to the TEE, ribosomes are rarely available to translate other mRNAs (4).
| + | <center><html><B>Fig. 1 Gene circuit of pVSW-3 series promoter reporting circuit </B></html></center> |
| − | ====<i>cspA</i> 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 <i>cspA</i> CRE because of the interaction between mRNA 5′-UTR and 3′-UTR.
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| − | | + | |
| − | ===Usage and design===
| + | |
| − | The XMU-China used pCspA, <i>cspA</i> 5′-UTR and 3′-UTR as cold-responsive elements (CRE), hoping to achieve the design of antifreeze proteins expressed at low temperature. We were inspired by the sequences of classic pCold series plasmids and introduced the TEE sequence to promote the binding of ribosomes to the mRNA. Here, we defined the promoter pCspA (<partinfo>BBa_K4907008</partinfo>), <i>cspA</i> 5′-UTR (<partinfo>BBa_K4907009</partinfo>), TEE sequence (<partinfo>BBa_K4907011</partinfo>) and <i>cspA</i> 3′-UTR (<partinfo>BBa_K4907010</partinfo>) together as the CspA cold-responsive expression cassette (CspA CREC), which allowed the insertion of CDS of target proteins into this cassette between TEE and <i>cspA</i> 3′-UTR (Fig. 1). Learn more from our Designs. | + | |
| − | Fig. 1回路图
| + | |
| − | | + | |
| | ===Characterization=== | | ===Characterization=== |
| − | ====Low-temperature induction==== | + | ====Agarose gel electrophoresis (AGE)==== |
| − | The low-temperature induction effect of CspA CREC was first characterized by GFP, which was regarded as the target protein and the corresponding CDS was designed to be inserted into the cassette. We directly synthesized the complete pCspA-<i>cspA</i> 5′-UTR-TEE-<i>gfp</i>-<i>cspA</i> 3′-UTR-B0015 on pSB1C3 vector (<partinfo>BBa_K4907118</partinfo>_pSB1C3). We transformed it directly into <i>E. coli</i> BL21(DE3) and verified by colony PCR (lane K4907118 in Fig. 2, target fragment-1546 bp).
| + | When building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1198 bp (lane K4907110). |
| − | Fig. 2胶图
| + | <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/bba-k4907110-p.png" width="400px"></html></center> |
| − | J23100-B0034-<i>gfp</i>-B0015_pSB1C3 (BBa_K4907146_pSB1C3, as a positive control group), BBa_I0500_pSB1C3 (as a negative control group) and the BBa_K4907118_pSB1C3 (as the experimental group) were characterized at 37 °C and 15 °C, respectively. As expected, the induction effect of low-temperature (15 °C) was obvious when GFP was expressed in CspA CREC, consistent with the increasing trend of reporter’s expression level at 15 °C as time progressed (Fig. 3b) but blunt changes at 37 °C (Fig. 3a). By contrast, when cultivated at 37 °C, the constitutively expressed GFP (under the control of J23100) showed a normal increasing trend of expression against time rather than a stagnant state at 15 °C, which might result from the negative influence of coldness to the bacteria. Based on this observation, although the well induction effect at 15 °C, means of alleviating the adverse impact of coldness or making the engineered bacteria more cold-adapted should be taken into account once the CspA CREC is applied at lower temperatures (such as 4 °C).
| + | <center><html><B>Fig. 2 The result of colony PCR. Plasmid BBa_K4907110_pSB3K3 </B></html></center> |
| − | | + | |
| − | Fig. 3 The comparison of normalized fluorescence intensity of different groups at (a) 37 °C and (b) 15 °C.
| + | |
| − | Therefore, in order to further characterize at lower temperatures, we introduced Mn-SOD (<partinfo>BBa_K4907132</partinfo>_pSB3K3) to enhance the stress resistance of the bacteria. This Mn-SOD-expressing plasmid and <partinfo>BBa_K4907118</partinfo>_pSB1C3 were co-transformed into <i>E. coli</i> BL21(DE3) and the correct dual-plasmid transformants were selected by chloramphenicol and kanamycin. The same characterization was performed at 4 °C. It can be seen from the Fig. 4 that CspA CREC still has much stronger expression strength under the condition of 4 °C, compared to the constitutively expressed J23100. Such results also showed the excellent performance of CspA CREC as a low-temperature induction expression system when accompanied with the expression of Mn-SOD.
| + | |
| − | | + | |
| − | Fig. 4 The comparison of normalized fluorescence intensity of different groups at 4 °C.
| + | |
| − | | + | |
| − | ====Leakage at high temperatures====
| + | |
| − | During the characterizations of different temperatures, a relative higher basal expression of CspA CREC was also observed, especially at 37 °C (Fig. 5), which indicated that there is an unneglected leakage of this cold-inducible expression system. For the purpose of a stringent control, we have come up with some means and improvements for lowering down the leakage of this CspA CREC system, resulting in various novel cold-inducible expression systems that would enrich the thermogenetic toolkits and contribute to other teams and labs (please see our Design page[https://2023.igem.wiki/xmu-china/design] for more information).
| + | |
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| − | Fig. 5 The comparison of normalized fluorescence intensity different groups at 37 °C for 6 hours. | + | ====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 <i>E. coli</i> 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). |
| | + | <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/biaozhen/xilieqidongzi19-16.png" width="300px"></html></center> |
| | + | <center><html><B>Fig. 3 The comparison of normalized fluorescence intensity the series promoters from pVSW-3(19) to pVSW-3(16). </B></html></center> |
| | + | ===Reference=== |
| | + | 1. S. Borukhov, E. Nudler, RNA polymerase: the vehicle of transcription. <i>Trends in Microbiology</i> <b>16</b>, 126-134 (2008). |
| | | | |
| − | Reference
| + | 2. H. Xia <i>et al.</i>, Psychrophilic phage VSW-3 RNA polymerase reduces both terminal and full-length dsRNA byproducts in in vitro transcription. <i>RNA Biology</i> <b>19</b>, 1130-1142 (2022). |
| − | 1. W. Bae, P. G. Jones, M. Inouye, CspA, the major cold shock protein of <i>Escherichia coli</i>, negatively regulates its own gene expression. <i>Journal of Bacteriology</i><b> 179</b>, 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. <i>Molecular Microbiology</i><b> 23</b>, 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. <i>Nucleic Acids Research</i> <b>43</b>, 6166-6179 (2015). | + | |
| − | 4. G. Qing et al., Cold-shock induced high-yield protein production in <i>Escherichia coli</i>. <i>Nature Biotechnology</i> <b>22</b>, 877-882 (2004).
| + | |
| | | | |
| | <!-- Add more about the biology of this part here | | <!-- Add more about the biology of this part here |
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(17) is one of them.
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(17) 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.
When building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1198 bp (lane K4907110).
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).
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).
