We wanted to test whether or not the amount of time we let the SCRIBE system run before inducing the system with ATC (to activate Cas 9 selection tool) made a difference in SCRIBE efficiency.

Total Plate Counts:

In this experiment, we want to see if running the SCRIBE system for 24 hours vs. 48 hours made a difference in the system efficiency when induced with ATC. We induced the cells containing cas9 and the SCRIBE system with ATC. Then, we plated these cells immediately on the 0 hour column to test the growth of the cells without the cas9 system have time to run. After one hour of the cells being induced with ATC, we plated them and repeated this procedure after two and three hours of ATC inducement.

The graph confirms that over time, SCRIBE works more efficiently. This bolsters the results from our rpoB experiment as the data were similar. According to our data, the amount of time we let the SCRIBE system run affected how well the cas9 enzyme was able to kill off the wildtype cells. The longer we ran the SCRIBE/cas9 system, (48 hours of SCRIBE) the lower the colony count was at after 3 hours when compared to the shorter run (24 hours of SCRIBE). This indicates that over time, the longer SCRIBE had to work on the system, the more mutants were made and later degraded by the CRISPR/cas9 system.

For the first part of our project, we are testing to see if Fahim Farzadfard and Timothy K. Lu’s paper, “Genomically encoded analog memory with precise in vivo DNA writing in living cell populations” rings true in regards to whether the SCRIBE system works or not. In order to check for the SCRIBE system, we incorporated a target sequence that gave rise to specific antibiotic resistance. rpoB gives rise to rifampicin resistance and rpsL gives rise to streptomycin resistance. If the SCRIBE system is working correctly, then the target sequence was successfully incorporated into the chromosomal DNA of the E.coli cell. Therefore the host cell will express the corresponding antibiotic resistance.

Rifampicin serial dilution plates

Experiments 1-4

Dilution factor: 10^-1 to 10^-3

Dilution factor: 10^-4 to 10^-6

We checked to see if the rpoB target sequence was incorporated into the pFF745 plasmid and whether it gave the host cells rifampicin resistance. This was proved by the growth of colonies on the rifampicin plates.

Experiment 1

The first experiment with E.coli represents the negative control therefore proving that pFF745 does not normally express rifampicin resistance.

Experiments 2, 3, and 4

Experiments 2, 3 and 4 are all E. coli with the rpoB target sequence. Since they were able to grow on the rifampicin plate, we can conclude the SCRIBE system successfully worked.

The sequencing data shows that the rpoB target was successfully put into the plasmid. The sequencing reaction is on the bottom and the designed plasmid sequence is on the top. The gray bars at the top show that the whole sequence matches.

Goal: We wanted to verify the host cells’ resistant to rifampicin antibiotic came solely from the rpoB mutation and not from another source.

rpoB is specific to rifampicin resistance while rspL is specific to streptomycin resistance. In order to rule out the possibility that other cells without the rpoB sequence can grow on rifampicin plates, we grew rpsL cells (with C and D orientation) on those plates. We patched the C and D cells on dilution plates, comparing cells that were and were not induced with IPTG (IPTG induces the SCRIBE system). As shown on the plate below, E. coli cells with rpsL as the target sequence were not able to grow on rifampicin plates. This supports our claim that only rpoB cells grew on the rifampicin plates.