Part:BBa_K4808000
Tcl40
In the biotechnological industry, temperature sensitive control systems has been widely employed, with the λpL/pR-cI system being the most common. One of its components, the pL/pR, is a temperature induced promoter originating from phage λ. Another component, cI, is a temperature sensitive repressor that can combine with the pL/pR promoter, and in result inhibiting the transcription process of the gene regulated by pL/pR promoter. Commonly, cI repressor forms a dimer and binds to pL/pR promoter, thereby inhibiting gene expression at low temperatures. Yet when temperatures rise to a certain point, the cI repressor shifts a dimer to two monomers, thus cancelling transcription inhibition. Clearly, the sensitivity to temperature of cI repressor is essential.
This year we improved BBa_C0051(wild type of cI from phage λ), Which we called TcI. We mutated TcI to create TcI40. We then tested the temperature induction threshold value of both TcI and TcI40. At a temperature of 40 degrees celcius, TcI40 loses its transcription inhibition effects, and reaches climax values of gene expression. We offer this part to future iGem teams needing a temperature sensitive control system.
Design
Figure: the state of cI repressor in different temperature
Characterization
To enable the practical large-scale implementation of our α-kb production method, we made the strategic decision to transition from using IPTG-induced expression to a more user-friendly and cost-effective temperature-controlled induction approach. To achieve this objective, we tested two temperature-sensitive repressors: TcI and TcI40.
In comparison to TcI, TcI40 exhibited nonsynonymous mutations at six distinct positions, resulting in alterations to six amino acids: L65S, A67T, K68R, F115L, D126G, and D188G. Additionally, seven synonymous mutations (base is changed but amino acid remains constant) were also present: A50, I69, E128, R129, T152, S160, and L185(fig.1B).
Figure 1: (A)The design of TcI-pR/pL control circuit. (B)The mutation of TcI40 compared to TcI.
We tested the two temperature sensitive repressors in the DH5a strain with RFP as the signaling protein, and the test was carried out in two groups each using one of the stated repressors. The first group's RFP was expressed using a plasimd containing repressor TcI, and the latter group used a plamid containing repressor TcI40. The visible red color of expressed RFP proteins indicates if or if not the repressors loses its inhibitive effects on transcription (and thus expression of genes) at a given temperature.
The results of this test show that at a temperature of 30 degrees Celsius, no visible red color can be seen (fig.2A), indicating that no RFP is expressed. Thus signaling that repressor TcI and TcI40 both show effective inhibition on the transcription process of RFP at such a given temperature. At a temperature of 37 degrees celcius, the transcription inhibition effect of repressor TcI ceases, and RFP expression values reaches its climax. At temperatures of 37, 40 and 42 degrees Celcius, the transcription inhibition effect of repressor TcI40 ceases, while climax values of RFP expression occur at a temperature of 40 degrees Celcius.
We have successfully tested two temperature control systems. By comparing TcI and TcI40 on RFP fluorescence at different induced temperatures, we can find that TcI40 can maximize the suppression of pR/pL promoter, so as to achieve the highest gene expression . In the future industrial production of a-KB, we hope to use TcI40 repressor to induce the production of a-KB to achieve temperature control production in large scale.
Figure2: Test TcI and TcI38 repressors at different induction temperatures.
References：
Cheng L, Wang J, Zhao X, et al. An antiphage Escherichia coli mutant for higher production of L-threonine obtained by atmospheric and room temperature plasma mutagenesis. Biotechnol Prog. 2020;36(6):e3058. doi:10.1002/btpr.3058 Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S. A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai). 2021;53(5):620-627. doi:10.1093/abbs/gmab036 Restrepo-Pineda S, O Pérez N, Valdez-Cruz NA, Trujillo-Roldán MA. Thermoinducible expression system for producing recombinant proteins in Escherichia coli: advances and insights. FEMS Microbiol Rev. 2021;45(6):fuab023. doi:10.1093/femsre/fuab023 Chen L, Chen Z, Zheng P, Sun J, Zeng AP. Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli. Appl Microbiol Biotechnol. 2013;97(7):2939-2949. doi:10.1007/s00253-012-4176-z Zhang C, Qi J, Li Y, et al. Production of α-ketobutyrate using engineered Escherichia coli via temperature shift. Biotechnol Bioeng. 2016;113(9):2054-2059. doi:10.1002/bit.25959 Park JH, Oh JE, Lee KH, Kim JY, Lee SY. Rational design of Escherichia coli for L-isoleucine production. ACS Synth Biol. 2012;1(11):532-540. doi:10.1021/sb300071a Hao R, Wang S, Jin X, Yang X, Qi Q, Liang Q. Dynamic and balanced regulation of the thrABC operon gene for efficient synthesis of L-threonine. Front Bioeng Biotechnol. 2023;11:1118948. Published 2023 Mar 2. doi:10.3389/fbioe.2023.1118948
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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