July Week1
Plasmid Amplification for further construction;
chose pRSFDuet-1, pACYCDuet-1 and pCDFDuet-1 for expression vector and pBluescript II KS(+) for connector.
July Week2
Construct the gene of our fusion protein: ssDsbA-FP-HL-Lgt-FL-TAL using splicing by overlap extension. We connected ssDsbA-FP-HL-lgt for one part of this fusion protein and FL-TAL for another. Then connect them together.
July Week3
Connected ssDsbA-FP-HL-lgt by splicing by overlap extension. Transform, colony picking plasmid extraction and digestion identification; Sequencing results showed accurate construction.
July Week4
Use golden gate to connected TAL of 2012 Freiburg igem. Transform, colony picking plasmid extraction and digestion identification.
August Week1-2
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 connected TAL in the right order. So we design some new primes 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.