Team:Florida/Improve
Improve a Previous Part or Previous Project
We improved Illinois's Part BBa_K1681001 (seen below) by adding a replacing pLacO with a stronger promoter called BBa_J23101 and adding the Van promotor in front of beta.
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.