Team:Cambridge/Notebook/Week5

Monday

We took photographs of the petri dishes that Theo and Peter had drawn. These had grown up well, over a short period. We believe this is because they were plated out with only ampicillin, and no chloramphenicol.

Will designed oligos to be sent to Biolegio for synthesis. Hannah and Emily wrote more messages on dishes, and planned a restriction digest to check for the biobrick compatibility of the V. fischeri lux operon.

Anja and Theo miniprepped an overnight culture which had been inoculated with a ligation of an expression backbone and firefly luciferase. They ran it on a gel, which showed that the insert had worked. Discovered that E-gels can be used multiple times. Then they performed a very bodged luciferase assay. 0.5ml of cells were frozen and thawed three times to lyse them, then 0.05ml of a concentrated D-luciferin solution was added. The camera showed them to be glowing.

Tuesday

Will constructed oligos, and taught Emily and Hannah to do so. V. phosphoreum arrived, Anja and Theo made up its specialised, salty culture. Peter and Theo placed it in five subcultures and inoculated.

Wednesday

Our brand new, fancy plate reader arrived on today from BMG for use to use for the next 2 months. We had a tutorial and are really looking forward to actually using it.

Emily and Hannah spent all day designing Phosphoreum oligos. It messed with our heads. Hannah became quite hysterical. Will designed oligos for site-directed mutagenesis of Phosphoreum. Emily sent off the V. fischeri oligo order.

Thursday

In the morning we had an e-mail saying we had slightly underestimated how much our oligo order would cost - Mike from Biolegio asked if we really wanted to spend £1200. Oops! So, we started re-thinking the order and deciding exactly which oligos we needed for the experiments we wanted to do. We also decided what we wanted to ask people about in the lab meeting in the afternoon.

In the afternoon, we had a lab meeting. We need to think more about what our 'big idea' is and plan out some more experiments.

Friday

Phosphoreum are glowing!

Will and Emily finished the re-designed V. fischeri oligos and sent them off to biolegio to be synthesised. Hannah started designing experiments to do with the oligos. Peter has been looking into oligos for quiescence. Bill has been transforming cells with the Pbad promoter from the registry. Will also grew up some cells so that we can extract plasmids to use oligos with.

Theo wrote a script to make the BMG plate reader take concurrent OD and luminescence reading over time, we set it going overnight. He also wrote a script in Python to process its output and spread some V. phosphoreum on plates. Theo also streaked out some lumazine (BBa_K216007) and luxAB (BBa_K216008)

Saturday

Theo and Peter came in, Theo confirmed that the script had measured OD successfully and set it going again with a longer timeframe. Luminescence results were unclear.

The V. phosphoreum had formed a thin film which was glowing brightly. Peter streaked it out in an attempt to get single colonies and did much needed tidying.

Theo examined the lumazine and luxAB colonies. Lumazine was unexpectedly pink, both were streaked out again to get better colonies. The plate reader was set running again for the rest of the weekend.