Part:BBa_K4907128

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

Biology

pCspA

pCspA (BBa_K4907008) is the promoter of CspA, which is a type of cold shock protein. 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 the 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 the CspA Cold-responsive Element (CRE) is not related to the pCspA, 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 the 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.

hrpR

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

Usage and design

If we use CspA Cold-responsive elements to express proteins at low temperatures, there will be a risk of gene leakage at a higher temperature than we expected because the response temperature 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, can the expression of downstream genes be induced, reducing the leaky expression.

Fig. 1 Gene circuits of hrp system regulated by the CspA CRE.

Characterization

Agarose gel electrophoresis (AGE)

When constructing this circuit of composite part BBa_K4907128 which includes the gene of HrpR, colony PCR and gene sequencing were used to verify that the transformants were correct. Target bands (1460 bp) can be observed at the position between 1000 and 2000 bp (Fig. 2).

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4907128_pSB1C3.

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 pHrpL will be activated when HrpR and HrpS are both expressed, but it will not be activated by HrpR or HrpS (BBa_K4907022) alone.

Fig. 3 The results of GFP Fluorescence/OD₆₀₀ to verify that hrp AND Gate can work.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]