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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 for 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 Week3：
There are some problems about Freiburg’s parts. We can’t connect TAL in the right order. So we design some new primers for PCR that can produce the right sequence.
August Week4
Design a few new ports for the fusion protein. Sequencing results showed accurate construction. Observe the FP using LSCM to confirm the fusion protein can locate on the membrane.
September Week1
Try co-transformation: Prsf pacyc pBluescript . Find the conditions of protein expression. Find the way to construct the TAL.
September Week2
Find the enzymes for the application. Find the way to detect the substrate in these pathways. Connector plasmid modification.
September Week3
TAL gene synthesis. Construct the part with our new ports.
September Week4
TAL gene synthesis.
