Team:Shenzhen/Result/YAO.Genome

Summary

Since our experimental time is limited, until now we only accomplished the second stage and design third stage. However, we are very luck to capture positive clones from the homologues recombination and shuttle plasmid experiments. Since it is still very difficult to find positive clones from shuttle plasmid experiments, we have not got good results for terminators test, and experiments in YAO.Sensor & YAO.Suicider. There are two possibilities for the failure in terminators test. One is no positive clones have been captured, and the other is that their termination efficiencies are too high that no green fluorescence could be captured.

Homologues Recombination

This is our BioBrick for first stage. Recombination sites have been chosen to be upstream and downstream sequences of cox3 gene in mtDNA.



Figure 1. BioBrick for homologues recombination experiment.

As showed in below, our gene gun based transformation system work and green fluorescence proteins spark in mitochondria. And no such signal can be tracked in control.



Figure 2. Homologues recombination experiment result, with positive clones on the left and controls on the right, under bright field and UV respectively.

Shuttle Plasmid

As first part succeeds, we design second part of YAO. Genome construction. Here are BioBricks we built for shuttle plasmids building. ori7 was selected to be the origin of replication site on plasmid, from NCBI database. About 500bp upstream sequence of cox1 gene, containing promoter and RBS, was chosen as regulatory.



Figure 3. BioBricks for shuttle plasmid experiment.

Shuttle plasmid was build in BioBricks standard and transformed into mitochondria.



Figure 4. Vector graph for our mtGFP expression shuttle plasmid.

To pick out positive clones is very hard and we luckily found one. Same as first stage experiment result, mtGFP expressed and no signal can be tracked in control.



Figure 5. Shuttle plasmid experiment result, with positive clones on the left and controls on the right, under bright field and UV respectively.

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Parts Annotation and Standardization

YAO.Genome Backbone