Team:Tsinghua/Result1

From 2009.igem.org

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==Top-Down Approach==
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The GenSniper genome synthesized by Top-Down approach only needs one plasmid. However, large DNA segment will be required for ligation, which will add difficulties to our experiment. The above picture is the map of the GenSniper genome with only one linkage group.
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Revision as of 05:10, 12 October 2009

Contents

Synthesis of the Genome of GenSniper

Bottom-Up Approach

j28

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.

j21

We have successfully cloned the Nu1-A, W-B, C and D-E-FI-FII gene segments from the bacteriophage lambda genome.

j22
j23

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.

j24

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.

j26

Enzyme digestion identification confirmed that the cloning of pACYCDuet-1+S+R+Rz+C+D+E+FI+FII is correct.

j27

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.

In summary, we have completed all the objectives in this section. The synthesized GenSniper genome should be functionally identified in the following experiments.

Top-Down Approach

j31

The GenSniper genome synthesized by Top-Down approach only needs one plasmid. However, large DNA segment will be required for ligation, which will add difficulties to our experiment. The above picture is the map of the GenSniper genome with only one linkage group.

j29
j30

GenSniper Production Evaluation

j32
j33
j34
j35
electro