Results
Control: Reporter plasmid in E.Coli - Will we be able to see fluorescence in cells with the reporter plasmid CFP.
Figure 1: Fluorescent images
We were able to show that E.coli transformed with the reporter plasmid expressed for the CFP gene as expected, although the level of fluorescence/expression was relatively low.
Experiment #1: Will SpCas9 + Reporter and SaCas9 + Reporter effectively degrade the Reporter plasmid, reducing CFP over time?
Unfortunately, due to our struggles with cloning and assembling our constructs, we were unable to perform any of the experiments we had planned, other than confirming expression of our reporter. Although we were able to see fluorescence in our reporter control through imaging a previous time, the control reporter during the experiments did not express enough CFP for comparison of time points and see further reduction.
We were unable to move forward with the rest of the experiments before the wiki freeze due to time constraints, issues with the double transformation required by the first experiment, and problems with SaCas9 and SpCas9 point mutagenesis to form respective dead Cas9s. We are continuing to work to complete this project and hope that we are able to present results from our planned experiments at the 2018 iGEM Jamboree.
Conclusion:
We were able to show fluorescence with the reporter plasmid; however, we did not have enough time to get any results from experiments. It is difficult to extrapolate meaningful results especially given the low level of CFP expression in the control, beyond basic observations that the reporter plasmid was successfully transformed and its reporter gene expressed to some degree. In the future, establishing a viable control may be facilitated through the use of competent cells which are optimized for protein expression (BL21), or by creating recombinant plasmids based on high-copy number vectors (although this would still have to be compatible multi-plasmid transformation). This would hopefully allow for more demonstrable and observable changes in CFP expression when cleaved by the “assassin” Cas9 protein, and serve as a baseline for fluorescence when blocked by the “guardian” Cas9.
Future Directions:
Given more time, we would establish controls that demonstrate baseline fluorescence, the ability of nuclease active Cas9 to cleave the target plasmid, and the ability of deactivated Cas9 (dCas9) to bind but not cut the target sequence. Once these controls are established and standardized, we would then move into the competition experiments. Working with the SaCas9 and SpCas9 systems we expect to see a considerable difference in the relative binding kinetics and affinity, giving us a clear winner in the competition. This data will then be incorporated into our mathematical modeling to show whether or not our predictions were correct and calculate a quantitative measure of relative binding affinity and kinetics that will be able to be compared between experiments. Following this process of validating the basic protocol, we will attempt to engineer a Cas9 enzyme to have some desired property and set up this experiment to compare the new design to the WT Cas9.
