Difference between revisions of "Part:BBa K4158010"

Latest revision as of 15:53, 12 October 2022

Psrtf1-GFP

This part encodes RBS, GFPuv coding site and a promoter regulated by SRTF1 and progesterone. SRTF1 is a transcriptional repressor specific to progesterone. Upon the binding of progesterone, GFP downstream of the binding region of SRTF1 is expressed as the repressor SRTF1 is deactivated. Thus, this part works as a reporter plasmid to confirm existence of progesterone.

Fig. 1. progesterone detector gene circuit

Design

Promoter and SRTF1 binding site: Sequence information was cited from a previous paper[1]. Consequently, the promoter sequence was the same as BBa_J23102 and the SRTF1 binding site was the same as BBa_K3889030.

RBS: We designed RBS by using RBS calculator and RNA fold as it optimizes to GFPuv.

Then, we constructed the plasmid by infusion cloning. A fragment of Psrtf1-gfp was inserted into pACYC184 vector which was cut on restriction enzyme sites of HindIII and BamHI.

Results

We demonstrated that this part could detect progesterone in the cell-free protein synthesis system in E.coli.

In order to detect progesterone in vitro, we transformed the SRTF1-expressing plasmid (BBa_K4158012) 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 performed cell-free protein synthesis reaction using the extracts and this reporter plasmid. Fig. 3. shows the result of the cell-free protein synthesis reaction. We added 100uM of progesterone in the final concentration.

From Fig. 3., we could confirm the following;

When progesterone was added in the absence of the reporter plasmid, an increase in GFP fluorescence was not observed (left).
When only the plasmid was added in the absence of progesterone, an increase in GFP fluorescence was slightly observed, due to the leak expression (middle).
When progesterone was added to the condition above, an increase in GFP fluorescence was observed (right).

Fig. 3. The result of progesterone detection by SRTF1

So, we concluded below.

Comparing the middle and the right, making activated SRTF1-enriched E.coli extract was successfully achieved.
Comparing the left and the right, engineering SRTF1(progesterone) regulated reporter plasmid(BBa_K4158010) was successfully achieved.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 13
Illegal NheI site found at 36
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 826
Illegal XhoI site found at 529
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

References

1. Sankar K et al. A progesterone biosensor derived from microbial screening. ACS Sens. 7(4):1132-1137(2022).

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4158010 short</partinfo>
-This part contains RBS, GFPuv coding site and a promoter regulated by SRTF1 and Progesterone((1S,3aS,3bS,9aR,9bS,11aS)-1-Acetyl-9a,11a-dimethyl-1,2,3,3a,3b,4,5,8,9,9a,9b,10,11,11a-tetradecahydro-7H-cyclopenta[a]phenanthren-7-one) and works as the reporter plasmid to confirm exist of Progesterone and SRTF1.
+This part encodes RBS, GFPuv coding site and a promoter regulated by SRTF1 and progesterone.
+SRTF1 is a transcriptional repressor specific to progesterone.
+Upon the binding of progesterone, GFP downstream of the binding region of SRTF1 is expressed as the repressor SRTF1 is deactivated.
+Thus, this part works as a reporter plasmid to confirm existence of progesterone.
-RBS+GFPuv sequence is the same as <partinfo>BBa_K4158011</partinfo>.
+[[File:Waseda Tokyo progesterone detector gene circuit.png|500px|thumb|center|Fig. 1. progesterone detector gene circuit]]
-We designed <b>Psrtf-GFP(<partinfo>BBa_K4158010</partinfo>)</b>, and confirmed its activity <i>in vitro</i>. This part is the reporter plasmid used for progesterone detection. It encodes GFP gene in the downstream of the binding site of SRTF1, the transcriptional factor specific to progesterone.
+<b>Design</b>
-Promoter and SRTF1 binding site: Sequence information was cited from paper[1].Consequently, the promoter sequence was the same as <partinfo>BBa_J32102</partsinfo> and the SRTF1 binding site was the same as BBa_K3889030.
+Promoter and SRTF1 binding site: Sequence information was cited from a previous paper[1]. Consequently, the promoter sequence was the same as <partinfo>BBa_J23102</partinfo> and the SRTF1 binding site was the same as <partinfo>BBa_K3889030</partinfo>.
-RBS: We designed the RBS by using RBS calculator and RNA fold as it optimizes to GFPuv.
+RBS: We designed RBS by using RBS calculator and RNA fold as it optimizes to GFPuv.
-Then, we constructed this part by infusion cloning. A fragment of Psrtf1-gfp was inserted into pACYC184 vector which was cut on restriction enzyme sites of HindIII and BamHI.
+Then, we constructed the plasmid by infusion cloning. A fragment of Psrtf1-gfp was inserted into pACYC184 vector which was cut on restriction enzyme sites of HindIII and BamHI.
-We demonstrated that this new part could detect progesterone in the cell-free protein synthesis system.
-[[File:Waseda Tokyo progesterone detector gene circuit.png|500px|thumb|center|Fig. 1. progesterone detector gene circuit]]
+<b>Results</b>
-[[File:Waseda Tokyo result of progesterone sensing by SRTF1.png|500px|thumb|center|Fig. 2. result of progesterone sensing by SRTF1]]
+We demonstrated that this part could detect progesterone in the cell-free protein synthesis system in <i>E.coli</i>.
-<b>Fig. 2.</b> shows the result of the cell-free protein synthesis reaction. All of the three samples contain the cell-free extracts expressing the transcription factor SRTF1. We added progesterone as 100uM in final concentration.
+In order to detect progesterone <i>in vitro</i>, we transformed the SRTF1-expressing plasmid (<partinfo>BBa_K4158012</partinfo>) into <i>BL21(DE3)Star</i> strain and prepared crude extracts which were pre-enriched with the transcription factor, SRTF1.
-We could confirm below from <b>Fig. 2.</b>.
+[[File:Waseda Tokyo Preparation of SRTF1-enriched extract.png|500px|thumb|center|Fig. 2. Preparation of SRTF1-enriched extract]]
-<ul>
+We performed <b>cell-free protein synthesis reaction</b> using the extracts and <b>this reporter plasmid</b>.
+<b>Fig. 3.</b> shows the result of the cell-free protein synthesis reaction. We added 100uM of progesterone in the final concentration.
+From <b>Fig. 3.</b>, we could confirm the following;
+<ul>
 <li>When progesterone was added in the absence of the reporter plasmid, an increase in GFP fluorescence was not observed (left).</li>
 <li>When only the plasmid was added in the absence of progesterone, an increase in GFP fluorescence was slightly observed, due to the leak expression (middle).</li>
 <li>When progesterone was added to the condition above, an increase in GFP fluorescence was observed (right).</li>
 </ul>
+[[File:Waseda Tokyo result of progesterone sensing by SRTF1.png|500px|thumb|center|Fig. 3. The result of progesterone detection by SRTF1]]
 So, we concluded below.
 <ul>
 <li>Comparing the middle and the right, making activated SRTF1-enriched E.coli extract was successfully achieved.</li>
-<li>Comparing the left and the right, <b>engineering SRTF1(progesterone) regulated reporter SRTF1 reporter gfp plasmid(<partinfo>BBa_K4158010</partinfo>) was successfully achieved</b>. we achieved a project based on BioBricks, which is an important standard component in synthetic biology.</li>
+<li>Comparing the left and the right, <b>engineering SRTF1(progesterone) regulated reporter plasmid(<partinfo>BBa_K4158010</partinfo>) was successfully achieved</b>.  </li>
 </ul>
-Thus, we succeeded in <b> engineering SRTF1 regulated gfp reporter plasmid(<partinfo>BBa_K4158010</partinfo>).</b>
-(For more details, go to https://2022.igem.wiki/waseda-tokyo/results#progesterone-detection.)
 <!-- Add more about the biology of this part here