We have successfully constructed our 12kbp plasmid and expressed the dCas9 protein. Here are the results:

1. Plasmid construction

After various restriction enzyme cuts and ligation, we have successfully constructed our 12kbp plasmid.

2. Successful Transformation

We have successfully transformed our plasmids into Microcystis and spread onto selective plates, showing that the plasmid we constructed is compatible in both E.coli and Microcystis. Besides, our transformed cells demonstrated kanamycin resistance. These are living colonies as we checked the plates under microscope every day. We ensured that the cells were not only dead cell bodies buy transfering them to new selective plates once in two weeks.

3. Plasmid expression

We used Microcystin-LR detection kit (WRZ001) from 天河綠洲. We believed that our dCas9-sgRNA complex has been successfully been expressed. This is because the Microcystin-LR concentration in our transformed Microcystis was lower than 0.002mg/L, while our control set ups have a higher concentration.

The successful transformation of Microcystis culture showed Microcystin concentration less than 0.002mg/L. This shows a decrease in Microcystin toxin.

4. Quantitative Real-Time PCR

We will be conducting quantitative real-time PCR in the future for further verification of our results.