"
Team:Tsinghua/Result1
From 2009.igem.org
Contents |
Synthesis of the Genome of GenSniper
Bottom-Up Approach
Showed above are the maps of the GenSniper using bottom-up approach. The genes of Nu1, A, W, B and lysis genes (S, R, Rz) are incorporated into pET-28a, while C, D, E, FI and FII are synthesized into pACYCDuet-1. In other word, the GenSniper will have two linkage groups.
We have successfully cloned the Nu1-A, W-B, C and D-E-FI-FII gene segments from the bacteriophage lambda genome.
Positive clones of the two linkage groups of the GenSniper genome have been achieved (termed pACYCDuet-1+C+D+E+FI+FII and pET-28a+Nu1+A+W+B). After PCR of the purified recombinant plasmids, we can get the desired bands on the gels. Nonspecific bands are due to the low annealing temperature.
After constructing the GenSniper genome with the structral proteins we need, we further incorporated the lysis genes (S+R+Rz) into pACYCDuet-1+C+D+E+FI+FII.
Enzyme digestion identification confirmed that the cloning of pACYCDuet-1+S+R+Rz+C+D+E+FI+FII is correct.
After constructing all the desired clones, we had to testing their compatibility. A shows the plate after cotransformation of pET-28a+Nu1+A+W+B and pACYCDuet-1+C+D+E+FI+FII. B shows plate after cotransformation of pET-28a+Nu1+A+W+B and pACYCDuet-1+S+R+Rz+C+D+E+FI+FII. The result clearly indicates that they can work together, which provides the basis for the functioning of the proteins encoded by the GenSniper genome via Bottom-Up synthesis.
Top-Down Approach
GenSniper Production Evaluation
