Aim 1
While Collin et al.’s (2021) switch-gRNA could identify trigger strands of mRNA, there was one critical issue: how to get it into the E. coli. This is Aim 1’s focus: to detect exogenous trigger RNA through transformations of E. coli. First, we decided to electroporate in trigger RNA to E. coli with a switch-gRNA and a CRISPR plasmid. If successful, CRISPR would target the bacteria’s antibiotic resistance, causing cell death. However, our results showed that there was little to no difference compared to control RNA. We believed this was due to the RNA degrading too rapidly, which led us to try RNase-resistant RNA.
One question remained, however: how much exogenous trigger RNA is necessary for detection when compared to a control? We tested different dilutions of RNA, ranging from a 1:1 to a 1:100 dilution, that was electroporated into E. coli with a switch-gRNA and CRISPR plasmid. If trigger RNA was used, we expected CRISPR to cut antibiotic resistance and cause cell death, while if control RNA was used, we expected no difference between any dilutions.
Reference the Experiments Page to view a complete list of the protocols used for each aim.
We found that RNase-resistant RNA worked, supporting the idea that it was due to degradation. However, when testing dilutions, we found that it was hard to see a few missing colonies among an entire plate of growth. This was due primarily to an ineffective output system, which prompted a shift in Aim 3 to create a negative selection system. Rather than look for a few missing colonies in tons of growth, this system would enable us to look for a few growing colonies among an entire plate of cell death, which is a much clearer output system. While we have yet to optimize dilutions due to obstacles in Aim 3, we have shown that electroporation of exogenous RNA into our engineered E. coli is successful. In plasma, the trigger RNA won’t be RNase-resistant, however, this shows that we could use RNase-deficient E. coli as a workaround to achieve similar results.
[Figure 1a] This diagram illustrates the transformation of RNA and ST plasmid into modified E. coli. Plasmid 1229 and an exogenous trigger RNA sequence (3) are transformed into E. coli, which results in a lack of growth due to Cas12a. We are testing the hypothesis that growth only occurs when the switch and trigger match.
[Figure 1b] These plate readings are a visual representation of the comparison of colony numbers between control and trigger RNAs. In this figure, bacteria with all 3 plasmids produce no colonies in the presence of a correct trigger RNA (1) and show growth in the presence of an incorrect trigger RNA (2). This allows us to confirm that growth only occurs when the switch and trigger match.