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Motivation
Motivation
Our experiment all started after a previous experiment due to its unexpected results. In the previous experiment they were testing to find the most effective spacer length to maximize the fluorescence produced by the Green Fluorescent Protein. The fluorescence for 17 base pair and 18 base pair spacers was expected to be the highest out of the lengths that they tested. But what wasn't expected was the spike in fluorescence with the 21 and 22 base pair samples.
The Plan
The Plan
We did this by mutating the spacer to prevent the possibility of binding earlier than anticipated in the spacer which would then prevent the increased fluorescence in these samples. We created very unfavorable binding conditions throughout the spacer which would ensure that binding would only occur where we wanted it to.
Final Spacer
Final Spacer
The red indicates where we predicted the polymerase was binding to allow for more fluorescence than we expected.
Controls
Controls
- Sigma Factors
- Testing Environment
- The Sig G
- The Original Test
Procedure
Procedure
Step 1:
Step 1:
We designed and copied a plasmid containing the mutated spacer
Step 2:
Step 2:
We prepared samples from the previous experiment and our new samples
Step 3:
Step 3:
We then tested these colonies fluorescence to see if our spacer was successful
Results
Results
Before Mutation
Before Mutation
This is a picture taken through a microscope from the 22 base pair sample from the orignal trial. It is clear that there is quite a lot of fluorescence present.
After Mutation
After Mutation
This is a picture taken through a microscope from the 22 base pair sample with the new mutated spacer. There is much less fluorescence.
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