Results
Projects
Cas-Find
Cas-Found
- IPTG inducible CRISPR-Cas9 guide RNA targeted to rRNA Reverse
- Investigated viable systems for future investigation
Can't-Find
- Use of vector (pET16B) with non-expected insert doomed 1/3 of our potential constructs
- Failure to clone Cas-LucC/N & Cas-LacZA/O into E.Coli
- As a result we have been unable to study the viablity of our proposed diagnostic tool
FUEL
Fuel Up
- We have successfully cloned our arabinose mkeima and Lux Operon-mKeima constructs
- We have demonstrated that the Lux Operon of the fusion construct is still functional
Running on Empty
- We have been unable to express mKeima from either of the constructs
- We have therefore been unable to observe any red shifting of the Lux Operon
- Again we have as a result of our issues with cloning, been unable to test our hypothesis.
There are allot of things that we have learnt from out first year and hopefully next year we will be in a much stronger position.
If we were to do this experiment again then:
We would likely use the pSB1C3 antibiotic resistance variants instead of the three distinct plasmids we chose to use (pSB1C3, pET16B, pCOLA-DUET). This would improve the control of copy number and therefore help uniform the expression of our constructs. P.S. it removes the chance of a mystery insert in one our plasmids.
We really struggled with our cloning with many of our constructs failing to transform. We thought it may potentially be due to the size's of our constructs, so next time we'd look at potentially using electrocompetent cells.
A number of our primers did not behave as expected and meant often we couldn't inspect our transformed cultures without directly sequencing them. Next time we will set more time and resources to checking all our primers work appropriately.
When we started lab work the whole team was involved. This has the effect of making the work a bit fractured and disjointed, not to mention complicating the management of samples. When we spoke to other teams it became apparent that it was the norm to have only a few members designated to lab duties. We therefore decided to reduce the number of us in the lab, and split off into two groups with each one focusing on a project each. This is something we look to do from the start next time.
We didn't start our FUEL project until after half of our Lab time was up, this really restricted the amount of time we could devote to it. Next time we would aim to split into two lab groups that start at the same together but work independently, which again would improve the management of lab work.
Collaborations
Provided Oxford with FLIM analysis of their copper chelators.
We have quantified ATP production from cells transformed with Washington's phosphoenolpyruvate kinase and phosphoglycerate kinase constructs
Unfortunately we were unable to practically collaborate with Sacley due to failures in our related dCas9. In future years we aim to increase our number of collaborations. We dedicated most of our lab time to cloning our own constructs, which meant there wasn't much time, or manpower left for collaborations.
