Team:Florida/Demonstrate
Demonstrate
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.
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