Ssbb14-Christopher J. Coates

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Chris Notebook

Christopher J. Coates 21:11, 13 February 2014 (EST)

Group conversation established good candidate organisms. Settled on Thermus thermophilus and Thermatoga maldima, although Aquivex aeolicus was considered until logistics prevented acquisition of genomic DNA.

Feb 13. 2014.

Assigned Arginine-aaRS and Serine-aaRS from Thermatoga maritima.

Overall run down of Experiment:

Feb 27. 2014. miniprep from cells pBad-*fill in this name* according to listed protocol.

Mar 4. 2014. PCR according to construction files for ArgRS and SerRS synthesis.

Mar 6. 2014.

Ran analytical gel on Pcr pdt / ladder and previously isolated vector [ladder, trp, tyr, ArgRS, SerRS, [] [] [] vector] Zymo clean up of ArgRS and SerRS bands. Everything looked good and clean.

Mar 18. 2014. Digested pcrpdt according to construction file using listed protocol.

Mar 20. 2014. Ran analytical gel ladder pBad pBad Arg Ser [] []. Everything looked clean.

Apr 3. 2014. Zymo clean Ligate Transform Plate

Apr 5. 2014. Zymo clean Ligate Transform Plate

Apr 8. 2014. Checked colonies, everything looked great. Picked 2 Arg and 2 Ser now labelled Arg1, Arg2, Ser1, Ser2. Inoculated picks for incubator and informed Danny to remove

Apr 10. 2014. Miniprep colonies Digest Analytical Gel Sequence Procedure

Apr 15. 2014. Pour gel (an ordeal) Digest (didn't happen)

Apr 17. 2014. Digest Arg products with BamH1 using listed protocol. Digest Ser products with EcoR1 using listed protocol. Run Gel Gel shows good banding for Ser, can send for sequencing. Ambiguous ghost banding for Arg, although it demonstrates the two expected digest bands on top of what was suggested to me to be uncut and monocut vector. Alternative mapping required. Miniprepped pBad

Apr 22. 2014. Digest Arg and Vect with alternative restriction enzyme BglII Analysis suggests that Ser1 is correct and Arg1 is probably correct

May 1. 2014. Run final gel to determine sequencing. Someone altered voltage to 180V and melted my gel mid run. No useful results. Submit samples based on previous evidence. Sequencing too late, never sent. However it seems clear from good colonies and clear early analytical gels that the part was transformed. And digestions partially confirm incorporation for Arg1 and definitely confirm for Ser1. Therefore the experiment has been reasonably carried.

There are a number of easy luciferase assays for ATP consumption available. It would have taken minimal resources to confirm activity of the t-RNA synthetases post boiling and this seems a logical next step. If these assays demonstrate little to no activity then the