Difference between revisions of "Part:BBa K3889021"
Line 7: Line 7:
 
[[File:T--IISER-Tirupati India--SRTF1 repression.png|centre]]
 
[[File:T--IISER-Tirupati India--SRTF1 repression.png|centre]]
  
−
Waseda_Tokyo 2022 performed some experiment with this parts.
 
  
−
We found out that BBa_K3889021 can't express in <i>E. coli</i> by SDS-page
+
<big><b>Contribution: Waseda_Tokyo 2022</b></big>
  
−
[[File:Waseda Tokyo result of SDS-PAGE SRTF1.png|500px|thumb|center|Fig. 1. result of SDS-PAGE (SRTF1)]]
+
Waseda_Tokyo 2022 used this part in the aim for detecting progesterone in cell-free protein synthesis (CFPS) system in <i>E.coli<i>.
 +
We demonstrated that this part did not work in <i>E.coli<i>. We transformed this plasmid into<i>E.coliBL21(DE3)Star</i> 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<i>E.coli</i> <partinfo>BBa_K4158012</partinfo> is shown as a control. As shown in Fig. 1., the molecular mass of the protein optimized for <i>Bacillus subtilis</i>was smaller than that of SRTF1 (22kDa). Thus, we found that SRTF1 gene optimized for <i>Bacillus subtilis</i> was not fully translated in<i>E.coli<i> due to the difference in codon usage in these two organisms.
  
−
SRTF is 22kDa.
 
−
<i>E. coli</i> codon optimized SRTF1(BBa_K4158012) seems that it expressed finely while <i>B. sub</i> optimized SRTF1(BBa K3889021) doesn't.
 
  
−
It is because BBa K3889021 contains <i>E. coli</i> rare codon, we guess.
+
[[File:Waseda Tokyo result of SDS-PAGE SRTF1.png|500px|thumb|center|Fig. 1. The result of SDS-PAGE (SRTF1)]]
 +
 
  
 
The nucleotide sequence of BBa K3889021(<i>B.subtilis</i> optimized SRTF1 gene) is shown below. <b>Bold</b> letters represent low-usage codons in <i>E.coli</i> [2]  
 
The nucleotide sequence of BBa K3889021(<i>B.subtilis</i> optimized SRTF1 gene) is shown below. <b>Bold</b> letters represent low-usage codons in <i>E.coli</i> [2]  

Revision as of 05:38, 10 October 2022


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]

T--IISER-Tirupati India--SRTF1 repression.png


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<i>. We demonstrated that this part did not work in <i>E.coli<i>. We transformed this plasmid into<i>E.coliBL21(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 forE.coli BBa_K4158012 is shown as a control. As shown in Fig. 1., the molecular mass of the protein optimized for Bacillus subtiliswas smaller than that of SRTF1 (22kDa). Thus, we found that SRTF1 gene optimized for Bacillus subtilis was not fully translated inE.coli<i> due to the difference in codon usage in these two organisms.


Fig. 1. The result of SDS-PAGE (SRTF1)


The nucleotide sequence of BBa K3889021(<i>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

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]


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).