IGEM:MIT/2006/Notebook/2007-6-29

=Things to Do Today=

1. Get streaked plates out of the warm room- DONE (all look good except J45120 & J45250 (no isolated colonies))

2. Make LCs of J45995, J45996, R0040.E0840, and B0015 for plate reader experiment tomorrow- DONE

3. Reserve plate reader for tomorrow- DONE

4. Research leucine pathway

http://jb.asm.org/cgi/reprint/136/1/1?view=long&pmid=361681, http://jb.asm.org/cgi/reprint/120/2/715.pdf, http://jb.asm.org/cgi/reprint/130/1/429?view=long&pmid=15983

Use of plasmids: http://aem.asm.org/cgi/reprint/63/12/4651?view=long&pmid=9406383, http://www.freepatentsonline.com/20040091980.html, It has now been found, surprisingly, that isolation of the ilvE gene present in E. coli ATCC 11303, and its cloning onto a multicopy plasmid, results, after transformation of the starting strain with this plasmid, in an increase of at least 3- to 5-fold in the yield of the particular amino acid- http://www.freepatentsonline.com/5120654.html

5. Email leucine calculations to Endy/Knight/grad students- DONE (Austin corrected my one calculation, we want 5 mM and the cells have ~.1 mM

6. Heard back from Yale stock center. Need to clarify what I heard on the phone in my conversation with Tribe. Send Tribe an email- DONE

7. Order a chorismate-overproducing strain from Yale- WILL HOLD OFF UNTIL I HEAR FROM TRIBE (looks like it may be trp-, tyr-, or AB3249

8. Restreak J45120 & J45250- DONE

9. HPLC isoamyl acetate with Andreas at neutral pH- DONE (DID NOT WORK, TOO MANY PEAKS, MUST LOOK INTO GC/MS OPTIONS)

10. Found another GC in the geobiology lab. Contact their lab technician inquiring about using the GC- DONE

11. Contact Professor Steinberg about enhancing leucine production in E. coli- DONE (Says he hasn't worked in this specific area since the '70's)

12. Had a long discussion with Professor Tribe. Settled on a strategy. I will order a strain that is aroF- aroG- aroH-. Then I can insert a G+ allele in via a plasmid and cotransform it with J45700 and J45800. This is "quick and dirty," but it should give us a chorismate overproducer. Contacted both Yale and one of Tribe's colleagues in Australia to find the necessary strain- DONE