Part:BBa_K3889021
Steroid Responsive Transcription Factor1 (SRTF1)
Steroid Responsive Transcription Factor (SRTF1) is a bacterial transcription factor that can respond to progesterone in a concentration-dependent manner. SRTF1 binds to its binding site (BBa_K3889030) resulting in inhibition of gene expression. Now as progesterone enters the system SRTF1 binds to progesterone leaving its binding site free for the transcription machinery to start working. This results in a progesterone inducible gene expression. [1]
Contribution: Waseda_Tokyo 2022
Waseda_Tokyo 2022 used this part in the aim for detecting progesterone in cell-free protein synthesis (CFPS) system in E.coli. We demonstrated that this part did not work in E.coli. We transformed this plasmid into E.coli BL21(DE3)Star strain, and made cell-free extracts enriched with the protein. Fig. 1 shows the result of SDS-PAGE performed with the bacterial extract. SRTF1 optimized for E.coli BBa_K4158012 is shown as a control. As shown in Fig. 1., the molecular mass of the protein optimized for Bacillus subtilis was smaller than that of SRTF1 (22kDa). Thus, we found that SRTF1 gene optimized for Bacillus subtilis was not fully translated in E.coli due to the difference in codon usage in these two organisms.
The nucleotide sequence of this part BBa K3889021 (B.subtilis optimized SRTF1 gene) is shown below. Bold letters represent low-usage codons in E.coli [2]
atg/tct/agt/act/gcg/gag/aga/ata/cgg/cca/gga/aga/agc/ggg/att/ctc/gct/gct/gcc/act/cgc/ctc/ttc/gcg/acg/cac/gga/gtg/tct/ggc/aca/tca/tta/caa/caa/ata/gcc/gat/gcc/act/ggt/atc/ aca/aaa/gct/gct/gta/tac/cat/cac/ttc/cct/acc/aaa/gaa/gaa/gtg/gtc/gtc/gct/gtc/ttg/gcg/cct/gcc/ctt/gag/gcc/atc/caa/ggc/atc/gtt/aga/aca/gca/ggg/gct/cac/gaa/gat/cct/cgc/gcc/ gct/acc/gag/gca/gcg/att/att/gga/ctc/gcg/gac/caa/gca/gtc/acg/cat/cgc/cag/cgc/tgg/gct/gtt/ctg/tta/caa/gat/gca/gca/gta/gaa/gaa/tac/gtc/cgt/aac/aat/cca/gac/cac/gat/gag/ttg/ ttc/act/cgt/ctg/aga/tta/ctg/ctt/act/ggc/ccc/gat/ccg/acc/cct/ggg/act/aga/ctg/caa/gtg/tcc/ctc/ttc/ttg/tct/ggt/ctt/ttg/ggg/cca/gca/caa/gat/cct/tct/tgc/gca/gat/ata/gac/gac/gat/gcg/ tta/aga/gcg/gga/atc/gta/cgt/gcg/ggc/cgt/aga/ctg/ctc/ttg/gcg/gat/gac/gac/gct/taa
We carried out experiments to detect progesterone in the E.coli cell-free system using cell-free extracts enriched with SRTF1, the progesterone-specific transcription factor. Initially, we performed CFPS experiments using existing iGEM part (https://parts.igem.org/Part:BBa_K3889100) which encodes CDS of SRTF1 optimized for expression in Bacillus subtilis. However, we could not observe its activity. SDS-PAGE of the extracts revealed that the molecular mass of the protein optimized for Bacillus subtiliswas smaller than that of SRTF1 (22kDa). This indicated that SRTF1 gene optimized for Bacillus subtilis was not fully translated inE.coli due to the difference in codon usage in these two organisms. Therefore, we optimized the CDS codon for E.coli and were able to detect progesterone in a cell-free system with this improved part. This part (https://parts.igem.org/Part:BBa_K4158012) is said to be an improvement of the existing part in that it allows SRTF1 to be expressed not only in Bacillus subtilis but also in the heterologous organism E.coli.
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]
References
1. Grazon, C., Baer, R. C., Kuzmanović, U., Nguyen, T., Chen, M., Zamani, M., Chern, M., Aquino, P., Zhang, X., Lecommandoux, S., Fan, A., Cabodi, M., Klapperich, C., Grinstaff, M. W., Dennis, A. M., & Galagan, J. E. (2020). A progesterone biosensor derived from microbial screening. In Nature Communications (Vol. 11, Issue 1). Springer Science and Business Media LLC. https://doi.org/10.1038/s41467-020-14942-5
2. Don Lorimer et al. Gene Composer: Database Software for Protein Construct Design, Codon Engineering, and Gene Synthesis. BMC Biotechnology 9(1):36(2009).
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