Team:Osaka/Notebook

Notebook

Week 1

Went through basic molecular biology lab skills and techniques with the new members. Extracted most of the existing parts required for our project from the iGEM 2011 Spring distribution plates, transformed and amplified the DNA for future use.

Week 2

DNA extraction & purification (miniprep) of last week's transformed parts, & gel runs to confirm lengths. Sequencer was broken so we could not confirm sequence, but most parts' lengths turned out ok. Ligations to put together some common combinations (eg promoter + RBS).

Week 3

Extraction & amplification of more basic parts from the distribution plates, plus ligation of a few more parts. Not much progress due to lab being closed for Obon holidays.

Week 4

Original plan was to use GFP as a reporter but spectrofluorometer was broken, so we amplified luciferase parts as a possible alternative. Tetracycline-resistance plasmid backbone was also amplified to enable more options during 3A assembly.

Week 5

Some of us attended iGEM Japan Meet-Up at Kyoto University. Did more ligations to start putting together lycopene biosynthesis gene cluster (CrtE, CrtB, CrtI) in case the 2009 Cambridge part BBa_K274100 doesn't work.

Week 6

Finally got our primers after 3 weeks' wait! PCR to amplify PprI, PprA, PprM and RecA from Deinococcus radiodurans genomic DNA. Preliminary trial of DNA damage detector using (E. coli RecA) promoter with lycopene biosynthesis (CrtEBI) as reporter. Results were a little disappointing, perhaps the RecA promoter doesn't work well in the δRecA strain that we used. Ordered a new batch of competent cells (different strain).

Week 7

Ligations of D. radiodurans genes to promoters, RBS, etc. Also, ligations to combine radioresistance parts: PprI-PprA, and PprI-PprA-PprM-PprM.

Week 8

Completion of radioresistance parts & drafting of radioresistance assay protocol.

Week 9

DNA repair/radioresistance assays: transformed cells were irradiated with UV light and then incubated on agar plates, and viability relative to control used as an evaluation of the abilities of PprI, PprA, PprM, RecA to confer resistance to radiation-induced DNA damage.

Week 10

Competent cells finally arrived, DNA damage detection assay! Also, wiki updating before the freeze!

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