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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.
