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. Start to synthesis the TAL gene

September Week2

Find the enzymes for the application. Find the way to detect the substrate in these pathways. Connector plasmid modification.

Start to design the primers used in the vector remoulding.

September Week3

The synthesized TAL gene sequence from a gene company received; continue to construct the part with our new ports.

remould the PSK vector in order to achieve our aims.

September Week4

Express the TAL gene;Do the test for prokaryotic expression.Express the constructed gene and get the final results.