The USC iGEM team is working to combat the fire blight plant pathogen, Erwinia amylovora, that ruins crops in apple and pear orchards along the West Coast. As a team located in Southern California, protecting our foliage is of the utmost importance as global warming continues to shift climate conditions.
We will be engineering the ɸ Ea104 bacteriophage, specific to E. amylovora, to attack the bacteria.
Our in vitro guide RNAs that direct Cas9 will cut the E. amylovora chromosome. Then this segment will undergo homologous recombination with a Bacterial Artificial Chromosome/Yeast Artificial Chromosome (BAC/YAC) cloning vector that has been designed with homologous ends. TAR cloning will be utilized to clone the viral bacteriophage genome into the yeast.
Once successfully cloned into the yeast nucleus, Cas9 editing strategies can be readily employed to modify the genome. Here, we will be able to delete a gene from the bacteriophage that is essential to replication for biocontainment purposes. Once this deletion is verified, we will move the cloned genome into E. coli for amplification and maintenance. In parallel, we will modify the E. amylovora genome to contain the gene which was deleted from the viral chromosome. Effectively, the modified viral chromosome will only be capable of replication in this strain. To test this, we will transform the modified viral chromosome into our modified E. amylovora strain and into a control wild-type strain to assay viral replication.