Team:Florida/Results
Results
Our overall project can be broken down into three components. (1) Verifying the SCRIBE system (2) Compare the efficiency of the SCRIBE system against using Cas9 as a selection tool (3) Using the Van promoter to make the SCRIBE system more efficient.
Verifying the SCRIBE 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
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.
Verifying Mutation is Caused by rpoB in SCRIBE only
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.
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.
rpoB1 plates over time
rpoB2 plates over time
rpoB3 plates over time
Efficiency of SCRIBE system vs. using Cas9 as a Selection Tool
Our team’s goal was to create a universal selection marker using the SCRIBE and CRISPR/Cas9 system. In this way, we can manipulate the SCRIBE system to incorporate any target sequence so that Cas9 can efficiently select for mutants that would otherwise be undetected by typical selection markers such as antibiotic resistance or color change.
Counting Colonies
SCRIBE without Cas9
SCRIBE+Cas9 Plate Counts
***Highlighted regions indicate outlier (not included in “Plate type vs. Colony Count (9/17) graph)***
We expected to see the plates that were induced with ATC (triple antibiotic and rifampicin plate) to have approximately the same amount of colony growth. This is because the triple antibiotic plate had only the cas9 as the selection tool while the induced rifampicin plate used both antibiotic resistance and the cas9 as selection tools. We believed that cas9 and antibiotic resistance would have similar efficiencies in killing wild type cells (non-mutants). According to our graph, we see that the cas9 and triple antibiotic plate had around the same number of colonies. Thus, we can support our hypothesis that cas9 works as a selection tool.
When we use Cas9 as a selection tool (as demonstrated by the triple antibiotic plate), we see that there are more colonies than on the induced rifampicin plate. Thus, it is possible that some wildtype colonies left on the plate when we use cas9 as opposed to using it in conjunction with rifampicin resistance (given by the SCRIBE system). This leads us to conclude that Cas9 can be used as a selection tool, but it is not as efficient when coupled with the SCRIBE system.
The uninduced LB and rifampicin plate did not contain ATC. Therefore, cas9 was not working on these plates. The LB plate showed significantly more colony growth since there was no selection present on the plate. Both wildtype and mutant cells were grown on LB plate. The rifampicin plate has fewer colonies growing because the antibiotic acts as the selection tool.
Van Promoter Increases Production of Beta Protein
When SCRIBE is turned on, it produces ssDNA. Beta proteins bind to the ends of the DNA, aiding in site specific homologous recombination of the sequence into the Okazaki fragments during lagging strand synthesis. They also protect ssDNA from degradation in the cell since the host cell would read the ssDNA as foreign DNA. Sometimes during transcription, the RNA polymerase will not reach the Beta subunit, leading to the early termination of transcription. This makes the system less than optimal because the beta subunit will not always be transcribed during replication. To prevent this, we incorporated the Van promoter to be in front of the B-subunit sequence, improving the overall efficiency of the SCRIBE system. Now, each component of the plasmid has its own promoter.
Experiments:
1. E. coli
a. Plated E. coli on LB and Rifampisin plates to act as negative control
2. Only SCRIBE system working (with Lac promoter)
a. Wanted to test for and see the current efficiency of the SCRIBE system
3. Lac promoter and van promoter
a. Testing to see if the van promoter make the SCRIBE system more efficient
4. J23 promoter and van promoter
a. Testing to see if replacing the Lac promoter with J23 promoter increased efficiency
b.Working alongside the van promoter
LB plates (Experiments 1-4)
Rifampicin plates (Experiments 1-4)
From this we can see that the E.coli was able to grow on the LB plates but not able to grow on the Rifampicin plates (Experiment 1). The SCRIBE system was more efficient when the van promoter was introduced into the system as evident from the difference in the colony counts for experiments 2 vs 3&4 (2 was only the SCRIBE system working while 3&4 had the van promoter working as well). We wanted to see if the J23 promoter worked better in place of the Lac promoter the J23 is a high efficiency promoter. On the rifampicin plate, we see that there was more colony growth with the J23 promoter than with Lac (Experiment 4 and Experiment 3 respectively). Both systems also had the van promoter in it which supports the claim that the van promoter increases SCRIBE efficiency.
Project Achievements
Successful Results
- Incorporating RpoB target sequence into SCRIBE
- Incorporating SCRIBE into DH5-alpha cells resistant to rifampicin
- Incorporating cas9 into pFF745
- Modifying pFF745 from chloramphenicol to kanamycin resistance
- Incorporating pkdtrpob565 into pFF745
- Incorporating SCRIBE, cas9, and pkdtrpob565 into DH5-alpha cells
- Using cas9 as a selection marker against wild type cells
- Incorporating J23101 promoter in front of reverse transcriptase
- Van promoter
- Van promoter was initially not successfully incorporated into the pFF745 plasmid
- To confirm this we sequenced the cells and found that the van promoter was not successfully incorporated into the plasmid.
- Therefore, we performed another transformation which successfully incorporated the Van promoter into the plasmid (PCR amplify the PvanCC promoter and the PFF backbone, and CPEC the two pieces together)
Unsuccessful Results
Column chromatography did not work when purifying DNA. Instead we used ethanol purification. This was an alternative DNA purification method to purify Van promoter because the column chromatography did not work.