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| - | <h2 id="July">July Week1: Plasmid Amplification</h2><br> | + | <h3 id="July">July Week 1: Plasmid Amplification</h3><br> |
| | <p>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.<p> | | <p>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.<p> |
| - | <h2 id ="JulyWeek2" >July Week2: Plan Making for Gene Construction</h2><br> | + | <img src="https://static.igem.org/mediawiki/2014/0/07/SJTU14-notebook-week1.jpg"width=50% height=50%> |
| - | <p>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 for another, so that they could be connected together.<p> | + | |
| - | <h2 id ="JulyWeek3" >July Week3: Part Construction</h2><br> | + | <h3 id ="JulyWeek2" >July Week 2: Plan Making</h3><br> |
| - | <p> 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.<p> | + | <p>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.<p> |
| - | <h2 id ="JulyWeek4" >July Week4</h2><br> | + | |
| - | <p> Use golden gate to connected TAL of 2012 Freiburg igem. | + | <h3 id ="JulyWeek3" >July Week 3: Construction of Part 1</h3><br> |
| - | Transform, colony picking plasmid extraction and digestion identification.<p> | + | <p> 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.<p> |
| - | <h2 id="August">August Week1-2</h2><br> | + | <img src="https://static.igem.org/mediawiki/2014/8/82/SJTU14-July_week3.jpg"width=50% height=50%> |
| - | <p>Test the conditions for the PCR. | + | <p><img src="https://static.igem.org/mediawiki/2014/8/83/SJTU14-July_week4.jpg"width=50% height=50%> |
| | + | |
| | + | <h3 id ="JulyWeek4" >July Week 4: TAL Connection</h3><br> |
| | + | <p> 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.<p> |
| | + | <img src="https://static.igem.org/mediawiki/2014/3/32/SJTU14-week4-1.jpg"width=10% height=10%> |
| | + | <img src="https://static.igem.org/mediawiki/2014/a/a3/SJTU14-week4-2.jpg"width=25% height=25%> |
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| | + | <h3 id="August">August Week 1-2: PCR Optimization</h3><br> |
| | + | <p>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.<p> | | Connected TAL, transform, colony picking plasmid extraction and digestion identification. Find with our electrophoresis band. Expression vectors and connector plasmid are confirmed by sequencing.<p> |
| - | <h2 id ="AugustWeek3" >August Week3</h2><br> | + | <img src="https://static.igem.org/mediawiki/2014/f/fb/SJTU14-August_week1~2-1.jpg"width=25% height=25%> |
| - | <p>There are some problems about Freiburg’s parts. We can’t connected TAL in the right order. | + | <img src="https://static.igem.org/mediawiki/2014/1/11/SJTU14-August_week1~2-2.jpg"width=35% height=35%> |
| - | So we design some new primes for PCR that can produce the right sequence.<p> | + | |
| - | <h2 id ="AugustWeek4" >August Week4</h2><br> | + | <h3 id ="AugustWeek3" >August Week 3：</h3><br> |
| - | <p>Design a few new ports for the fusion protein. | + | <p>There are some problems about Freiburg’s parts. We can’t connect TAL in the right order. |
| - | Sequencing results showed accurate construction. Observe the FP using LSCM to confirm the fusion protein can locate on the membrane.<p> | + | So we designed some new primers for PCR that could produce the right sequence.<p> |
| - | <h2 id="September" >September Week1</h2><br> | + | |
| - | <p>Try co-transformation: Prsf pacyc pBluescript . | + | <h3 id ="AugustWeek4" >August Week 4</h3><br> |
| - | Find the conditions of protein expression.
| + | <p>We designed a few new adaptors for the fusion protein. |
| - | Find the way to construct the TAL.<p> | + | Sequencing results showed accurate construction. Then we observed the FP using LSCM to confirm that the fusion protein could locate on the membrane.<p> |
| - | <h2 id ="SeptemberWeek2" >September Week2</h2><br> | + | |
| - | <p>Find the enzymes for the application. | + | <h3 id="September" >September Week 1</h3><br> |
| - | Find the way to detect the substrate in these pathways.
| + | <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> |
| - | Connector plasmid modification.<p> | + | |
| - | <h2 id ="SeptemberWeek3" >September Week3</h2><br> | + | <h3 id ="SeptemberWeek2" >September Week 2</h3><br> |
| - | <p>TAL gene synthesis. Construct the part with our new ports.<p> | + | <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> |
| - | <h2 id ="SeptemberWeek4" >September Week4</h2><br> | + | |
| - | <p> TAL gene synthesis.<p> | + | <h3 id ="SeptemberWeek3" >September Week 3</h3><br> |
| | + | <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 ="SeptemberWeek4" >September Week 4</h3><br> |
| | + | <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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| | </article> | | </article> |
| | </div> | | </div> |
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