Difference between revisions of "Team:HUST-China/Part Collection"

 
(4 intermediate revisions by the same user not shown)
Line 262: Line 262:
 
     <div class="container-fluid member-head">
 
     <div class="container-fluid member-head">
 
         <div class="container">
 
         <div class="container">
−
             <h2 class="text-center">Part collection</h2>
+
             <h2 class="text-center">Part Collection</h2>
 
             <p style="color:#fff;margin-bottom:10px;text-align:center;">Following documented composite parts are respectively from three circuits this year:</p>
 
             <p style="color:#fff;margin-bottom:10px;text-align:center;">Following documented composite parts are respectively from three circuits this year:</p>
 
         </div>
 
         </div>
Line 382: Line 382:
 
<p>
 
<p>
 
We used the same method as Cro and pRE characterization to test the inhibition of CI and pR. From the figure above we can clearly see that CI does reduce the expression of GFP. </p>
 
We used the same method as Cro and pRE characterization to test the inhibition of CI and pR. From the figure above we can clearly see that CI does reduce the expression of GFP. </p>
−
<p>And we also did Fluorescence microscope detection after 30, 120 and 240 minutes induction.</p>
+
<p>And we also did fluorescence microscope detection after 30, 120 and 240 minutes induction.</p>
 
<img src="https://static.igem.org/mediawiki/2016/6/63/T--HUST-China--Experiments-CI-pR_Flou-detec.png" alt="">
 
<img src="https://static.igem.org/mediawiki/2016/6/63/T--HUST-China--Experiments-CI-pR_Flou-detec.png" alt="">
−
<p>From pictures above,the fluorescence of both two groups was increasing over time and it is obvious that the test group which contains CI expressed less GFP protein than control group. The results verify the inhibition of CI to pR from a more intuitive way.</p>
+
<p>From pictures above,the fluorescence of both two groups was increasing over time and it is obvious that the test group which contains CI expressed less GFP protein than control group. The results verify the inhibition of CI from pR in a more intuitive way.</p>
 
<h2>CII and pRE</h2>
 
<h2>CII and pRE</h2>
 
<p>3. CII and pRE activation test</p>
 
<p>3. CII and pRE activation test</p>
Line 396: Line 396:
 
   <td><a href="http://parts.igem.org/Part:BBa_K2036011"> BBa_K2036011 </a></td>
 
   <td><a href="http://parts.igem.org/Part:BBa_K2036011"> BBa_K2036011 </a></td>
 
   <td> pRE-GFP-LVAssrAtag</td>
 
   <td> pRE-GFP-LVAssrAtag</td>
−
   <td> A control group of CII and pRE interaction</td>
+
   <td> A control group of CII and pRE interaction.</td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
Line 407: Line 407:
 
<img src="https://static.igem.org/mediawiki/2016/e/ef/T--HUST-China--CII-pRE_plate.png" alt="">
 
<img src="https://static.igem.org/mediawiki/2016/e/ef/T--HUST-China--CII-pRE_plate.png" alt="">
 
<img src="https://static.igem.org/mediawiki/2016/4/4b/T--HUST-China--Experiments-CII-pRE_Flou-detec.png" alt="">
 
<img src="https://static.igem.org/mediawiki/2016/4/4b/T--HUST-China--Experiments-CII-pRE_Flou-detec.png" alt="">
−
<p>The method of verifying CI and pR inhibition is also applied to test activiation of CII to pRE.From line chart and fluorescence detection,we can see that the test group contains CII expressed more GFP protein than control group.</p>
+
<p>The method of verifying CI and pR inhibition is also applied to test activiation of CII to pRE.From line chart and fluorescence detection,we can see that the test group containing CII expressed more GFP protein than control group.</p>
 
<h2>CIII and Ftsh</h2>
 
<h2>CIII and Ftsh</h2>
 
<p>4. CIII and Ftsh</p>
 
<p>4. CIII and Ftsh</p>
Line 429: Line 429:
 
<img src="https://static.igem.org/mediawiki/2016/f/f9/T--HUST-China--Experiments-Fig6.png" alt="">
 
<img src="https://static.igem.org/mediawiki/2016/f/f9/T--HUST-China--Experiments-Fig6.png" alt="">
 
<p>Moreover,other characterization circuit in the collection can also serve as an individual part to construct positive and negative control of interest genes.</p>
 
<p>Moreover,other characterization circuit in the collection can also serve as an individual part to construct positive and negative control of interest genes.</p>
−
<h2>Tri-stable switch</h2>
+
<h2>Tri-stable Switch</h2>
−
<p>The final circuit can serve as a signal Filter, and users just need to add two sensors and two interest genes into the circuit. We highly recommend to use our submission plasmid as template and employ In-Fusion methods to achieve the assembly.</p>
+
<p>The final circuit can serve as a signal filter, and users just need to add two sensors and two interest genes into the circuit. We highly recommend to use our submission plasmid as template and employ In-Fusion methods to achieve the assembly.</p>
−
<p>To test the switch fetures, we build a characterizationi circuit with GFP and RFP as reporter genes, ptrp and pT7 as sensors:</p>
+
<p>To test the switch fetures, we build a characterization circuit with GFP and RFP as reporter genes, ptrp and pT7 as sensors:</p>
 
<table border="1">
 
<table border="1">
 
<tr>
 
<tr>
Line 450: Line 450:
 
                 <img src="https://static.igem.org/mediawiki/2016/4/46/T--HUST-China--Experiments-Fig14-3.png" alt="">
 
                 <img src="https://static.igem.org/mediawiki/2016/4/46/T--HUST-China--Experiments-Fig14-3.png" alt="">
 
                 <p>In ideal conditions, RFP is supposed to be visualized through fluorescence microscope when induced by IPTG. And after adding IAA into the medium, our host strain will turn to the other state of GFP expression. And because of LVAssrA tag’s effect, the RFP to GFP expression transition will be relatively obvious.</p>
 
                 <p>In ideal conditions, RFP is supposed to be visualized through fluorescence microscope when induced by IPTG. And after adding IAA into the medium, our host strain will turn to the other state of GFP expression. And because of LVAssrA tag’s effect, the RFP to GFP expression transition will be relatively obvious.</p>
−
                 <h2>Application cicuit</h2>
+
                 <h2>Application Circuit</h2>
−
                 <p>To provide an application example, we constructed an application circuit based on this version to help relief lactose intolerance (<a href="https://2016.igem.org/Team:HUST-China/Demonstrate">more details see to application page</a>) .
+
                 <p>To provide an application example, we constructed an application circuit based on this version to help relieve lactose intolerance (<a href="https://2016.igem.org/Team:HUST-China/Demonstrate">more details see to application page</a>) .
 
                 </p>
 
                 </p>
−
                <img src="https://static.igem.org/mediawiki/2016/a/a3/T--HUST-China--modeling-application.png" alt="">
+
                                <table border="1">
−
                <table border="1">
+
 
                     <tr>
 
                     <tr>
 
                         <td>Part Number</td>
 
                         <td>Part Number</td>
Line 466: Line 465:
 
                     </tr>
 
                     </tr>
 
                 </table>
 
                 </table>
−
                 <p>In the bi-stable switch of prokaryotes, we set the gene of interest 1 as iLDH and gene of interest 2 as beta-galactosidase. For sensing the signal, we choose lactose inducible promoter plac(mutant) and base inducible promoter patp2 (works when the pH of microenvironment is about 7~9).</p>
+
                 <p>In the bi-stable switch of prokaryotes, we set the gene of interest 1 as iLDH and gene of interest 2 as beta-galactosidase. For sensing the signal, we choose lactose-inducible promoter plac(mutant) and base-inducible promoter patp2 (works when the pH of microenvironment is about 7~9).</p>
 
                 <img src="https://static.igem.org/mediawiki/2016/a/a3/T--HUST-China--modeling-application.png" alt="">
 
                 <img src="https://static.igem.org/mediawiki/2016/a/a3/T--HUST-China--modeling-application.png" alt="">
−
                 <p> And we hope more team to join and add more possibilities to the circuit!</p>
+
                 <p> And we hope more teams to join and add more possibilities to the circuit!</p>
 
             </article>
 
             </article>
 
         </div>
 
         </div>

Latest revision as of 19:52, 19 October 2016

Parts

Part Collection

Following documented composite parts are respectively from three circuits this year:

Collection List

Here is the list of our part collection:

Part Number Name Description
BBa_K2036009 pRM-GFP-LVAssrAtag A control group of Cro and pRM interaction characterization.
BBa_K2036010 Cro-TT-pRM-RBS-GFP-LVAssrAtag To test Cro and pRM interaction intensity.
BBa_K2036011 pRE-GFP-LVAssrAtag A control group of CII and pRE interaction.
BBa_K2036013 RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag To characterize CII’s interaction with pRE.
BBa_K2036014 RBS-CIII-RBS-CIII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag To characterize CIII’s interaction with Ftsh.
BBa_K2036015 RBS-CII-RBS-CII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag Tandem of CII amplifies its function.
BBa_K2036016 pR-GFP-LVAssrAtag A control group of CI and pR interaction.
BBa_K2036017 CI-TT-pR-RBS-GFP-LVAssrAtag To characterize CI and pR interaction.
BBa_K2036027 pRE-RBS-Cro-RBS-CII-TT-ptrp-RBS-CI-TT-pR-RBS-CIII-RBS-RFP-LAAssrAtag-TT-pRM-RBS-GFP-LVAssrAtag To verify the function of the whole circuit with T7 and ptrp sensing the signals.
BBa_K2036028 placm-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-ompA-iLDH-TT-pRM-RBS-beta-galactosidase To verify bi-stable function of eukaryotic version of signal filter.

The part collection well documents the prokaryote regulation circuit of our project with every detailed characterization of the transcriptionally related proteins and promoters.

The whole circuit employs bacteriaphage lambda operon and is redesigned as a positive-feedback tri-stable switch. Users can define two inputs and three kinds of outputs to make a sense-response system or an analysis-report system.

To characterize the internal protein-protein and protein-promoter reactions, we build four sets of test circuits:

Cro and pRM

1.Cro and pRM inhibition test:

Part Number Name Description
BBa_K2036009 pRM-GFP-LVAssrAtag A control group of Cro and pRM interaction characterization
BBa_K2036010 Cro-TT-pRM-RBS-GFP-LVAssrAtag To test Cro and pRM interaction intensity.

The control group and test group are seperately shown as below:

We inserted the circuit into PET-Duet-1 palsmid and used plate reader to see the flourescence difference.

We can see from the figure above that the flourescence of test group went down and seperated clearly from the control.

CI and pR

2.CI and pR inhibition test

Part Number Name Description
BBa_K2036016 pR-GFP-LVAssrAtag A control group of CI and pR interaction.
BBa_K2036017 CI-TT-pR-RBS-GFP-LVAssrAtag To characterize CI and pR interaction.

We used the same method as Cro and pRE characterization to test the inhibition of CI and pR. From the figure above we can clearly see that CI does reduce the expression of GFP.

And we also did fluorescence microscope detection after 30, 120 and 240 minutes induction.

From pictures above,the fluorescence of both two groups was increasing over time and it is obvious that the test group which contains CI expressed less GFP protein than control group. The results verify the inhibition of CI from pR in a more intuitive way.

CII and pRE

3. CII and pRE activation test

Part Number Name Description
BBa_K2036011 pRE-GFP-LVAssrAtag A control group of CII and pRE interaction.
BBa_K2036013 RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag To characterize CII’s interaction with pRE.

The method of verifying CI and pR inhibition is also applied to test activiation of CII to pRE.From line chart and fluorescence detection,we can see that the test group containing CII expressed more GFP protein than control group.

CIII and Ftsh

4. CIII and Ftsh

Part Number Name Description
BBa_K2036014 RBS-CIII-RBS-CIII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag To characterize CIII’s interaction with Ftsh.
BBa_K2036015 RBS-CII-RBS-CII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag Tandem of CII amplifies its function.

Moreover,other characterization circuit in the collection can also serve as an individual part to construct positive and negative control of interest genes.

Tri-stable Switch

The final circuit can serve as a signal filter, and users just need to add two sensors and two interest genes into the circuit. We highly recommend to use our submission plasmid as template and employ In-Fusion methods to achieve the assembly.

To test the switch fetures, we build a characterization circuit with GFP and RFP as reporter genes, ptrp and pT7 as sensors:

Part Number Name Description
BBa_K2036027 pRE-RBS-Cro-RBS-CII-TT-ptrp-RBS-CI-TT-pR-RBS-CIII-RBS-RFP-LAAssrAtag-TT-pRM-RBS-GFP-LVAssrAtag To verify the function of the whole circuit with T7 and ptrp sensing the signals.

In ideal conditions, RFP is supposed to be visualized through fluorescence microscope when induced by IPTG. And after adding IAA into the medium, our host strain will turn to the other state of GFP expression. And because of LVAssrA tag’s effect, the RFP to GFP expression transition will be relatively obvious.

Application Circuit

To provide an application example, we constructed an application circuit based on this version to help relieve lactose intolerance (more details see to application page) .

Part Number Name Description
BBa_K2036028 placm-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-ompA-iLDH-TT-pRM-RBS-beta-galactosidase To verify bi-stable function of eukaryotic version of signal filter.

In the bi-stable switch of prokaryotes, we set the gene of interest 1 as iLDH and gene of interest 2 as beta-galactosidase. For sensing the signal, we choose lactose-inducible promoter plac(mutant) and base-inducible promoter patp2 (works when the pH of microenvironment is about 7~9).

And we hope more teams to join and add more possibilities to the circuit!