Difference between revisions of "Part:BBa K3889021"

Latest revision as of 14:19, 11 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. 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.

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

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

Parts Improvement: Waseda_Tokyo 2022

BBa_K4158012

This part contains RBS and SRTF1-SSGSSG-TEV-His coding site. SRTF1 is a bacterial(steroid metabolizing bacterium; Pimelobacter simplex) transcription factor.

SRTF1 sequence is optimized to E. coli codon. This part is the improvement of BBa_K3889021(B. sub optimized SRTF1).

Fig. 1. progesterone detector gene circuit

Design SRTF1 amino acid sequence was cited from a previous paper[1].

We designed this part by

optimizing SRTF1 amino sequences to E.coli codon by using GeneArt(Thermo).
optimizing the 5'UTR by using RBS calculator and RNA fold.

Then, we inserted the fragment into pET26b(+) vector.

Results

In order to detect progesterone in vitro, we transformed this part into BL21(DE3)Star strain and prepared crude extracts which were pre-enriched with the transcription factor, SRTF1.

Fig. 2. Preparation of SRTF1-enriched extract

We also prepared cell-free extract enriched with the B. subtilis optimized SRTF1, using BBa_K3889021. Then, we performed SDS-PAGE to compare the expression of the protein in E. coli with both of the parts.

From Fig. 3., we could confirm that SRTF1(22kDa) codon-optimized for E.coli was successfully expressed in E. coli. On the other hand, 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 due to the difference in codon usage in these two organisms.

Fig. 3. The result of SDS-PAGE (SRTF1)

We also performed cell-free protein synthesis reaction with the extracts, using BBa_K4158010 as the reporter plasmid. Fig. 4. shows the result of the experiment. We added 100uM of progesterone in the final concentration. We demonstrated that this part could detect progesterone in the cell-free protein synthesis system.

Fig. 4. comparison of result of progesterone sensing by SRTF1

We could confirm below from Fig. 4..

The plain(BBa_K4158012): there is a significant difference of the fluorescence between progeterone added and not added.
The shaded(BBa_K3889021): there isn't a significant difference of the fluorescence between progeterone added and not added..

Thus, we succeeded in engineering SRTF1-expressing plasmid which works in E.coli, as a part improvement of BBa_K3889021. 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

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).

@@ Line 7: / Line 7: @@
 [[File:T--IISER-Tirupati India--SRTF1 repression.png|centre]]
-We found out that BBa_K3889021 can't express in <i>E. coli</i> by SDS-page
-[[File:Waseda Tokyo result of SDS-PAGE SRTF1.png|500px|thumb|center|Fig. 1. result of SDS-PAGE (SRTF1)]]
+<big><b>Contribution: Waseda_Tokyo 2022</b></big>
-SRTF is 22kDa.
+Waseda_Tokyo 2022 used this part in the aim for detecting progesterone in cell-free protein synthesis (CFPS) system in <i>E.coli</i>.
-E. coli codon optimized SRTF1(BBa_K4158012) seems that it expressed finely while B. sub optimized SRTF1(BBa K3889021) doesnt't.
+We demonstrated that this part did not work in <i>E.coli</i>. We transformed this plasmid into <i>E.coli BL21(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.
-It is because BBa K3889021 contains <i>E. coli</i> rare codon, we guess.
-The nucleotide sequence of BBa K3889021(<i>B.subtilis</i> optimized SRTF1 gene [2]) is shown in the below. <b>Bold</b> letters represent low-usage codons in <i>E.coli</i> [3]
+[[File:Waseda Tokyo result of SDS-PAGE SRTF1.png|500px|thumb|center|Fig. 1. The result of SDS-PAGE (SRTF1)]]
+The nucleotide sequence of this part 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]
 atg/tct/<b>agt</b>/act/gcg/gag/aga/ata/<b>cgg</b>/cca/<b>gga</b>/aga/agc/<b>ggg</b>/att/ctc/gct/gct/gcc/act/cgc/ctc/ttc/gcg/acg/cac/<b>gga</b>/gtg/tct/ggc/aca/tca/tta/caa/caa/ata/gcc/gat/gcc/act/ggt/atc/
@@ Line 23: / Line 24: @@
 ttc/act/cgt/ctg/aga/tta/ctg/ctt/act/ggc/<b>ccc</b>/gat/ccg/acc/cct/<b>ggg</b>/act/aga/ctg/caa/gtg/tcc/ctc/ttc/ttg/tct/ggt/ctt/ttg/<b>ggg</b>/cca/gca/caa/gat/cct/tct/tgc/gca/gat/ata/gac/gac/gat/gcg/
 tta/aga/gcg/<b>gga</b>/atc/gta/cgt/gcg/ggc/cgt/aga/ctg/ctc/ttg/gcg/gat/gac/gac/gct/taa
+<big><b>Parts Improvement: Waseda_Tokyo 2022</b></big>
+<big><partinfo>BBa_K4158012</partinfo></big>
+This part contains RBS and SRTF1-SSGSSG-TEV-His coding site.
+SRTF1 is a bacterial(steroid metabolizing bacterium; <i>Pimelobacter simplex</i>) transcription factor.
+<u><b>SRTF1 sequence is optimized to <i>E. coli</i> codon.
+This part is the improvement of <partinfo>BBa_K3889021</partinfo>(<i>B. sub</i> optimized SRTF1)</b></u>.
+[[File:Waseda Tokyo progesterone detector gene circuit.png|500px|thumb|center|Fig. 1. progesterone detector gene circuit]]
+<b>Design</b>
+SRTF1 amino acid sequence was cited from a previous paper[1].
+We designed this part by
+<ul>
+<li>optimizing SRTF1 amino sequences to <i>E.coli</i> codon by using GeneArt(Thermo).</li>
+<li>optimizing the 5'UTR by using RBS calculator and RNA fold.</li>
+</ul>
+Then, we inserted the fragment into pET26b(+) vector.
+<b>Results</b>
+In order to detect progesterone <i>in vitro</i>, we transformed this part into <i>BL21(DE3)Star</i> strain and prepared crude extracts which were pre-enriched with the transcription factor, SRTF1.
+[[File:Waseda Tokyo Preparation of SRTF1-enriched extract.png|500px|thumb|center|Fig. 2. Preparation of SRTF1-enriched extract]]
+We also prepared cell-free extract enriched with the <i>B. subtilis</i> optimized SRTF1, using <partinfo>BBa_K3889021</partinfo>.
+Then, we performed SDS-PAGE to compare the expression of the protein in E. coli with both of the parts.
+From <b>Fig. 3.</b>, we could confirm that SRTF1(22kDa) codon-optimized for <i>E.coli</i> was successfully expressed in <i>E. coli</i>.
+On the other hand, 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.
+[[File:Waseda Tokyo result of SDS-PAGE SRTF1.png|500px|thumb|center|Fig. 3. The result of SDS-PAGE (SRTF1)]]
+We also performed cell-free protein synthesis reaction with the extracts, using <partinfo>BBa_K4158010</partinfo> as the reporter plasmid.
+<b>Fig. 4.</b> shows the result of the experiment. We added 100uM of progesterone in the final concentration.
+We demonstrated that this part could detect progesterone in the cell-free protein synthesis system.
+[[File:Waseda Tokyo comparison of result of progesterone sensing by SRTF1.png|500px|thumb|center|Fig. 4. comparison of result of progesterone sensing by SRTF1]]
+We could confirm below from <b>Fig. 4.</b>.
+<ul>
+<li>The plain(<partinfo>BBa_K4158012</partinfo>): there is a significant difference of the fluorescence between progeterone added and not added.</li>
+<li>The shaded(<partinfo>BBa_K3889021</partinfo>): there isn't a significant difference of the fluorescence between progeterone added and not added..</li>
+</ul>
+Thus, <b>we succeeded in engineering SRTF1-expressing plasmid which works in <i>E.coli</i>, as a part improvement of <partinfo>BBa_K3889021</partinfo></b>.
+<partinfo>BBa_K4158012</partinfo> is said to be an improvement of the existing part in that it allows SRTF1 to be expressed not only in <i>Bacillus subtilis</i> but also in the heterologous organism <i>E.coli</i>.
 <!-- Add more about the biology of this part here
@@ Line 41: / Line 100: @@
 ===References===
 . 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
-. Don Lorimer et al. Gene Composer: Database Software for Protein Construct Design, Codon Engineering, and Gene Synthesis. <i>BMC Biotechnology</i> <b>9</b>(1):36(2009).
-. Raymond, A., Lovell, S., Lorimer, D. et al. Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer. <i> BMC Biotechnol</i> <b>9</b>, 37 (2009).
+. Don Lorimer et al. Gene Composer: Database Software for Protein Construct Design, Codon Engineering, and Gene Synthesis. <i>BMC Biotechnology</i> <b>9</b>(1):36(2009).