Target Plasmid Construction

One of our goals is to revolutionize the CRISPR system by making it more efficient; this will be undergone by designing one plasmid that can target multiple RNAs inside an organism. The purpose of the target plasmid is to test our CRISPR system. We can configure our CRISPR plasmid so that it destroys both the GFP and RFP RNA products produced by the target, and then prove by quantifying the fluorescence that our CRISPR system can destroy multiple RNAs at once.

Our target plasmid has both a GFP and an RFP coding sequence, as well as resistance to kanamycin. It has an origin of replication that will be compatible with the plasmid containing the CRISPR system so that the two plasmids can exist together in an E. coli cell.

We made the target plasmid by removing an RFP stuffer from plasmid pSB1C3 284, and then ligating that RFP stuffer into plasmid pSB3k3 332 that contains GFP, the appropriate antibiotic resistance, and the correct origin of replication for E.coli. We had the most trouble with the ligation of the RFP stuffer into the new GFP plasmid. After we cut out the RFP stuffer with restriction enzymes Xba1 and Pst1, we ran the product on a gel and gel extracted the 1kB RFP fragment. One major issue was that an inaccurate DNA ladder was being run on the gel. Our fragments did not appear at the correct sizes, so our solution was to extract all fragments, and separately ligate a sample of each fragment size into their own tube of parent GFP plasmid, with the expectation that one of these constructs is our target and will be verified to emit green and red fluorescent light when undergoing flow cytometry analysis.

Another change we made was to the plasmid containing GFP, kanamycin resistance, and the origin of replication (p15?). The original plasmid we used had the SarA promoter before the GFP coding sequence. We changed the plasmid so the promoter was (what was it?), a promoter that is native to E.coli.

To test if our transformation of the GFP-RFP target plasmid into E.coli worked, we did a flow cytometry analysis. Flow cytometry is used to separate cells by detecting fluorescent markers on cells. Since GFP and RFP naturally fluoresce, a liquid culture of the transformed colonies was grown and analyzed by the flow cytometer. The cells fluoresced both green and red, indicating that our target plasmid successfully transformed into E.coli cells and can express two functional fluorescent proteins.

This experiment was repeated to create a cell stock of the target and to confirm that the 1kb fragment of the plasmid with the RFP stuffer contained the RFP coding sequence. After digesting and running the 1kb fragment of the plasmid on an agarose gel, the 1kb fragment was gel extracted and ligated into our GFP host plasmid. During this verification task, the greatest obstacle was in attaining an effective transformation back into E. coli to verify our results. Finally, the newly ligated target plasmid was proven to have entered E. coli and have functional fluorescent proteins within the cell.

The target plasmid was verified using gel diagnosis, flow cytometry, and sequencing to ensure the actual length of fragments matched those predicted, ensure functionality, and to make certain that the plasmid was created as expected.