Team:SJTU-BioX-Shanghai/daynotes

From 2014.igem.org

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                     <h3 id ="AugustWeek3" >August Week3:</h3><br>
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                     <h3 id ="AugustWeek3" >August Week 3:</h3><br>
                     <p>There are some problems about Freiburg’s parts. We can’t connect TAL in the right order.  
                     <p>There are some problems about Freiburg’s parts. We can’t connect TAL in the right order.  
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                           So we design some new primers for PCR that can produce the right sequence.<p>
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                           So we designed some new primers for PCR that could produce the right sequence.<p>
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                     <h3 id ="AugustWeek4" >August Week4</h3><br>
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                     <h3 id ="AugustWeek4" >August Week 4</h3><br>
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                     <p>Design a few new ports for the fusion protein.  
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                     <p>We designed a few new adaptors for the fusion protein.  
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                           Sequencing results showed accurate construction. Observe the FP using LSCM to confirm the fusion protein can locate on the membrane.<p>     
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                           Sequencing results showed accurate construction. Then we observed the FP using LSCM to confirm that the fusion protein could locate on the membrane.<p>     
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                     <h3  id="September" >September Week1</h3><br>
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                     <h3  id="September" >September Week 1</h3><br>
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                     <p>Try co-transformation: Prsf  pacyc  pBluescript .  
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                     <p>We tried co-transformation: pRSF, pACYC and pBluescript. Also, we found the conditions of protein expression. What's more, we were still trying to find the way to construct the TAL. Meanwhile, we were starting to synthesis the TAL gene.<p>         
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                          Find the conditions of protein expression.  
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                          Find the way to construct the TAL.
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                          Start to synthesis the TAL gene <p>         
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                     <h3 id ="SeptemberWeek2" >September Week2</h3><br>
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                     <h3 id ="SeptemberWeek2" >September Week 2</h3><br>
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                     <p>Find the enzymes for the application.
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                     <p>With the current experimental results, we were beginning to find the enzymes for the application and the way to detect the substrate in these pathways. In addition, we did some modification to connector plasmids.<p>     
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                          Find the way to detect the substrate in these pathways.  
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                          Connector plasmid modification.<p>     
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                    <h3 id ="SeptemberWeek3" >September Week 3</h3><br>
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                     <p>Start to design the primers used in the vector remoulding.
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                     <p>We finally received the synthesized TAL gene sequence from the gene company, so we continued to construct the part with our new adaptors. In addition, PSK vector was remoulded in order to achieve our aims.</p>
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                     <h3 id ="SeptemberWeek3" >September Week3</h3><br>
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                     <h3 id ="SeptemberWeek4" >September Week 4</h3><br>
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                     <p>The synthesized TAL gene sequence from a gene company received; continue to construct the part with our new ports.<p>
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                     <p>We began to express the TAL gene and do some tests for prokaryotic expression. Constructed gene were expressed and the final results were obtained.<p>     
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                    <p>remould the PSK vector in order to achieve our aims.
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                    <h3 id ="SeptemberWeek4" >September Week4</h3><br>
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                    <p>Express the TAL gene;Do the test for prokaryotic expression.Express the constructed gene and get the final results.<p>     
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                 </article>
                 </article>

Latest revision as of 23:59, 17 October 2014

Week Notes
Protocol

July Week 1: Plasmid Amplification


We chose pRSFDuet-1, pACYCDuet-1 and pCDFDuet-1 as expression vectors and pBluescript II KS(+) as the connector. These plasmids were amplified for further construction.

July Week 2: Plan Making


We intended to construct the gene of our fusion protein, ssDsbA-mRFP-HL-Lgt-FL-TAL-His Tag, using overlap PCR, enzyme digestion and ligation. After careful consideration, we decided to connect ssDsbA-mRFP-HL-Lgt as one part of this fusion protein and FL-TAL-His Tag as another, so that they could be connected together.

July Week 3: Construction of Part 1


We constructed the first part, ssDsbA-mRFP-HL-Lgt with overlap PCR and ligated it into the pBluescript II KS(+). Then we obtained more plasmids through transformation, colony picking and plasmid extraction. After that, we verified them with digestion identification and sequencing. Sequencing results showed accurate construction.

July Week 4: TAL Connection


In order to construct the second part, we had to obtain the TAL we needed using bioparts from 2012 Freiburg iGEM team. But unfortunately, we didn't get any positive result.

August Week 1-2: PCR Optimization


Because of the negative results, we decided to adjust some PCR parameters, including the annealing temperature, template concentration and cycle number. Test the conditions for the PCR. Connected TAL, transform, colony picking plasmid extraction and digestion identification. Find with our electrophoresis band. Expression vectors and connector plasmid are confirmed by sequencing.

August Week 3:


There are some problems about Freiburg’s parts. We can’t connect TAL in the right order. So we designed some new primers for PCR that could produce the right sequence.

August Week 4


We designed a few new adaptors for the fusion protein. Sequencing results showed accurate construction. Then we observed the FP using LSCM to confirm that the fusion protein could locate on the membrane.

September Week 1


We tried co-transformation: pRSF, pACYC and pBluescript. Also, we found the conditions of protein expression. What's more, we were still trying to find the way to construct the TAL. Meanwhile, we were starting to synthesis the TAL gene.

September Week 2


With the current experimental results, we were beginning to find the enzymes for the application and the way to detect the substrate in these pathways. In addition, we did some modification to connector plasmids.

September Week 3


We finally received the synthesized TAL gene sequence from the gene company, so we continued to construct the part with our new adaptors. In addition, PSK vector was remoulded in order to achieve our aims.

September Week 4


We began to express the TAL gene and do some tests for prokaryotic expression. Constructed gene were expressed and the final results were obtained.