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Week 12: Monday 27th September- Sunday 3rd October
We had begun to collaborate with the UNAM-Genomics_Mexico team, they have catalogued our communications in detail. We had given them advice on protocols but they were not having any more success so at their request Peter sent them DNA for 5 constructs, luxCDABE and Luciferase and LREs from both Luciola cruciata and Photinus pyralis with and without promoters.
Paul's plate reader experiments continued to give somewhat unexpected results, with an increase in light emission which seemed to be correlated with entry into stationary phase. We wanted to know if this was some strange promoter effect or a feature of the luciferase pathway. For these purposes Emily and Bill attempted to assemble fluorescent proteins both under pBAD alone and under pBAD with the rest of the operon. Paul set up another plate reader experiment with a wide range of arabinose concentrations used for induction. Will performed further site-directed mutagenesis of LC luciferase, in case Ben's failed.
Emily and Bill continued trying to assembly the fluorescent proteins with pBAD and PP luciferase. The process proved more difficult than we had hoped, but we got there eventually.
Theo also continued experimenting with his electrical engineering skills with the E.glometer: trying to build a DIY light measurement device.
In the evening we made a live recording of the Gibson Assembly Song at Bill's so that we could record the music video in the lab tomorrow.
Paul continued with his plate reader experiments, this time comparing the light output from all the different L. cruciata luciferase colour mutants.
The transformations from the fluorescent proteins showed some very small colonies. We left them to grow for longer.
We had a lot of fun recording the Gibson Assembly Song video in the lab today. Theo has very good camera skills.
Today there were decent sized colonies on the fluorescent protein plates. However, it looked like there was a lot of cross-contamination - red colonies on lots of different plates that shouldn't have RFP. Very confusing. After examining the plates we just took colonies that appeared to be excited at the correct wavelengths and glow the right colour.
Ben, Emily and Bill recorded the individual tracks for the Gibson Assembly Song which Bill mixed using Garage band. It's slowly coming together.
Aware that next week we would need to pack up the lab to move into Jim Ajioka's lab in parasitology next week, we started slowly tidying up the lab :( It was a sad time indeed. Especially when the motivational posters of Anja had to come down off the walls.
We also realised clearing up the lab might take a while.
Week 12: Monday 27th September - Sunday 3rd October
114. Expt: Continuation of cultures for Mexico (Peter)
Sent to Mexico using Interparcel/DHL.
Tracking number 903532038716
Paid for by Peter: £26.50
115. Expt: Biobrick assembly of fluorescent proteins (YFP, CFP, RFP) into PP+pBAD+pSB1C3 and pBAD+pSB1C3 (Emily and Bill)
pSB1C3 from freezer was used at 12.9ng/µl
See continuation below.
116. Expt: Plate reader readings for Arabinose induction for LCLuc+PPLuc under pBAD
Arabinose concentration varying from 0µM to 10mM
D-luciferin at 100µM -> 15µl.
Total volume 100µl per well. 60.5µl of overnight culture
117. Expt: Repeat of Ben's experiment of altering the other 4 sites of LC luciferase using directed mutagenesis (Will)
PCR mixes were:
Did not work - abandon experiment
118. Continuation of Biobrick assembly of fluorescent proteins (YFP, RFP, CFP) into PP+pBAD+pSB1C3 and pBAD+pSB1C3 (Emily)
Followed protocol on p91 using following quantities:
119. Expt: Plate reader of: