Team:Cambridge/Notebook/Week2

Monday

In the morning we did further research into the enzymes required for luciferase recovery. We found that the product of the reaction is CHBT, a substrate which can be converted into luciferin but is much cheaper. You can see the pretty picture of the luciferase cycle here.

In the afternoon we presented our proposals to our supervisors who said we could look at trying to optimise luciferase and LRE for E. coli (perhaps using mutagenesis), we shouldn't assume that anything with bacterial luminescence will work (after all, this is biology) and we should think about chassis design. They also thought our idea of trying to make different colours would be very cool.

We also received a new, broken toy of a low light camera, which Bill and Theo tried to get working. After receiving an e-mail at about 4pm saying we needed to give a 15 minute presentation the next day in Newcastle, Hannah and Anja started working on that too.

Tuesday

• 06.50 - Train to Newcastle for UK iGEM get-togther

Wednesday

Thursday

• 11am - Health and Safety talk by Barbara
• Working lunch at the Waffle Company. Summary of discussion:
  • To do:
    • Ordering
    • Getting in touch with MIT (turning off lights)
    • T-Shirts
    • Project Plan (Gantt Chart etc)
  • To do long-term:
    • Modelling
    • Firefly Bioluminescence
    • Bacterial Bioluminescence
    • Human Practices
    • Quiescence
  • Individual roles:
    • Outline of experiments, protocols - Anja, Ben, Peter (especially controls)
    • Modelling - Paul, Bill, Emily
    • COSHH forms - Bill
    • LRE biobrick - Theo
    • Researching Bacterial Lux operon - Will, Hannah

Friday

Meeting with Laura Rowe in the Biotechnology Dept. at 3pm:

• Brightness measurements are integrated over time, so when comparing them make sure they are integrated over a long time (otherwise you are only measuring how quickly luminescence occurs, not how bright).
• Different colours:
  • Could use fluorophores instead of fluorescent proteins but need to ensure they attach to protein (could be a project in itself). Requires resonance energy transfer.
  • Using mutant luciferases probably easier to implement.
• Expression in E. coli: inclusion bodies could be a problem when trying to over-express genes. To test: break open cells and centrifuge twice (at a higher speed second time around), if pellets are formed these are probably inclusion bodies. Could then dissolve these with solvent and test for bioluminescence from proteins within inclusion bodies.
• Relative light units are used because different camera properties, distances etc. all mean that photons/sec are relative. Can use a source for calibration - tritium standards are used (e.g. MGM instruments).

Paper from Duncan about mutant bacterial strain with very bright luminescence to be investigated further.

Saturday

Sunday