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. MCC109 was the Rho0 strain (without mtDNA), and GW22 and DBY947 mutants were selected to be Rho+ strains (with mtDNA), with lysine-deficient.



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. This experiment is based on Rho+ strain Y187, with ura-deficient.



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.

Parts Annotation and Standardization

• Promoters

    The promoter sequences for YAO project are found in literatures:



Table 1. Promoters sequences for yeast mitochondial DNA. ( Deshpande, A. P. and S. S. Patel (2012). "Mechanism of transcription initiation by the yeast mitochondrial RNA polymerase." Biochim Biophys Acta.)

• RBS

The RBS sequence for mitochondria is discerned from the mito-chromosome based on following principles:

 - 1) RBS are on up-stream of CDS sequence and down-stream of promoter sequence in 5’-UTR sequence.

 - 2) The ribosome binds on RBS sequence and starts translation when coming across the first AUG codon.

    So the sequence between the last ATG codon and CDS on the DNA sequence is believed to contain RBS. With further modifications, we made those sequences BioBricks.

• Terminators

    Mitochondria transcription systems are similar to bacterial transcription system and the RNA polymerase in yeast mitochondria is very similar in sequence and structure to bacteriophage T7 RNA polymerase. With DNA secondary structure analysis of 5’UTR sequence of mitochondrial genes, we found the stem-loop structure in these sequences which are very similar to secondary structure of bacterial Rho-independent terminator. So we synthesized these sequences with some modifications to make them BioBricks and tested these terminators in our experiments. ( Deshpande, A. P. and S. S. Patel (2012). "Mechanism of transcription initiation by the yeast mitochondrial RNA polymerase." Biochim Biophys Acta.)



Figure 6. Secondary structures for annotated terminators for 1) cox3, 2) cob, 3) q0255.

    BioBricks have been constructed to test the termination efficiency. For we have just one reporter in mitochondrial codon table (mtGFP), we directly put the terminator in the middle of regulatory and mtGFP. As the terminator works, green fluorescence should be limited. However, we failed to pick up positive clones in this experiments. 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. If we construct another reporter that can be used in mitochondria, we will redesign this experiments using C.Dog protocol.



Figure 7. Terminator test BioBricks.

YAO.Genome Backbone

    We also minimized the mitochondrial genome, with the annotation database. Deleting all know coding sequences, reserving replication related, translational and transcriptional elements, and non-studied sequences, to be the YAO.Genome backbone.

File:Result yao genome p9.jpg

Figure 8. YAO.Genome backbone illustration.

    This figure below shows our image of our YAO. Genome which will have all features as mentioned above. Segments with different colors represent different genes. These genes constructed different systems. Different systems in YAO. Genome will provide clues to improve producing efficiency, purity, safety and to further research about nucleus and mitochondria co-regulation. Maybe we can achieve it in the near future.

File:Result yao genome p10.jpg

Figure 9. YAO.Genome imagination.