Difference between revisions of "Part:BBa K4907127"

Revision as of 14:32, 11 October 2023

pCspA-cspA 5'-UTR-TEE-hrpS-cspA 3'-UTR-B0015

Biology

P_cspA

P_cspA (BBa_K4907008) is the promoter of CspA, which is a type of cold shock proteins. When E. coli is transferred from 37 ℃ to 15 ℃, 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 temperature (1).

cspA 5’UTR

5’ UTR refers to the untranslated region at 5’ end of mRNA. As for cspA 5’ UTR(BBa_K4907009), 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, but the 5' UTR plays a greater role in the induction of downstream genes due to its conformational change (3).

TEE

TEE(BBa_K4907011) 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

3’ UTR refers to the untranslated region at 3’ end of mRNA. The stability of cspA 3’ UTR(BBa_K4907010) has been shown to play a major role in cspA CRE because of the interaction between mRNA 5' UTR and 3' UTR.

hrpS

This part(BBa_K4907022) codes for HrpS protein. HrpS protein binds to HrpR protein coded by hrpR (BBa_K4907021) forming a complex and then triggering the transcription of hrpL promoter(BBa_K4907019) (5).

Usage and design

If we use CspA Cold-responsive elements to express proteins at low temperature, there will be a risk of gene leakage at a higher temperature than we expected because the response temperature of it has a broad range. To solve this problem, we plan to combine a logic AND gate with the CspA CRE. Based on it, we designed an AND gate to respond to low temperature, namely, hrp AND gate. In this system, the hrpR and hrpS genes are regulated by the CspA CRE (Fig. 1). Under low-temperature conditions, only when both proteins are expressed, then can the expression of downstream genes be induced, reducing the leaky expression.

Characterization

Agarose gel electrophoresis (AGE)

When constructing this circuit of composite part BBa_K4907127 which includes gene of HrpS, colony PCR and gene sequencing were used to verify that the transformants were correct. Target bands (1424 bp) can be observed at the position between 1000 and 2000 bp (Fig. 2).

Dual-plasmid system transformation

We used dual-plasmid system transformation to prove the hrp AND gate. One control and three experimental groups were set up. For R+S group, Plasmid BBa_K4907123_pSB3K3 and plasmid BBa_K4907126_pSB1C3 were transformed into E. coli DH10β which can express HrpR and HrpS. For the remaining two experimental groups, each can only express one of HrpR (BBa_K4907021) and HrpS (BBa_K4907022). As for the control, Plasmid BBa_BBa_K4907123_pSB3K3 and plasmid BBa_BBa_I0500_pSB1C3 were transformed into E. coli DH10β. The positive transformants were selected by kanamycin and chloramphenicol.

Fluorescence measurement

Colonies harboring the correct plasmid were cultivated and induced. The expression behavior of GFP is observed by measuring the GFP Fluorescence/OD600 using microplate reader (Fig. 3).The results of fluorescence showed that the P_hrpL will be activated when HrpR and HrpS are both expressed, but it will not be activated by HrpR or HrpS (BBa_K4907022) alone.

Reference

1. W. Bae, P. G. Jones, M. Inouye, CspA, the major cold shock protein of Escherichia coli, negatively regulates its own gene expression. J. Bacteriol. 179, 7081-7088 (1997).
2. L. Fang, W. N. Jiang, W. H. Bae, M. Inouye, Promoter-independent cold-shock induction of cspA and its derepression at 37 degrees C by mRNA stabilization. Mol. Microbiol. 23, 355-364 (1997).
3. K. Yamanaka, M. Mitta, M. Inouye, Mutation analysis of the 5′ untranslated region of the cold shock cspA mRNA of Escherichia coli. J. Bacteriol. 181, 6284-6291 (1999).
4. G. L. Qing et al., Cold-shock induced high-yield protein production in Escherichia coli. Nat. Biotechnol. 22, 877-882 (2004).
5. 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