1. I had to stop the time course yesterday at 13 hours. I think I will shake the flasks at 110 RPM this time because I felt the cultures were growing too quickly.
2. I've gotten suggestions from both Pittard and Tribe about the mutations necessary to make excess chorismate. Both involve ordering strains from Yale. I will look into this today. Still in communication with them.
3. The J45900 and J45600 cultures are still growing with 15 mM of Leucine in the media- J45600 did not grow up (lethal amount of leucine?), J45900 did not smell :(, wonder if cells just have a hard time picking up leucine
Things to Do Today
1. Run the GC/MS of the 9- and 13-hour samples to see how effective the 1 hour pulse-chase time is- showed up although not too high
2. Do a new time course experiment with both J45120 and J45181 cultures, shaking them at 110 RPM- DOING
3. Do the C18 Column Reverse Chromatography with Sauer lab- DIDN'T WORK (Andreas says we'll try again when it's free)
4. Look at relevant article http://pubs.acs.org/cgi-bin/article.cgi/jafcau/2002/50/i14/pdf/jf0115645.pdf
5. Look at mutations for Pittard/Tribe--> aroG- TA mutation at 22, making Leu8 Ile8 and CT mutation making Ser180 Phe 180 http://jb.oxfordjournals.org/cgi/content/abstract/116/5/986, aroF- Asn8 to Lys8 or deletion of 7 N-terminal amino acids http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T2W-43PGJY7-V&_user=501045&_coverDate=08%2F07%2F2001&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000022659&_version=1&_urlVersion=0&_userid=501045&md5=5c9268b4066b39a2821532bf86f49456
1:03- J45120 time course starts, 2:03- take out J45120 with salicylic acid added, 5:03- add salycilic acid to a culture, 6:03- extract that culture, 9:03- add salicylic acid to a culture, 10:03- extract that culture, 1:03- add salicylic acid to a culture, 2:03- extract that culture, 5:03- add salicylic acid to a culture, 6:03- extract that culture, 9:03- add salicylic acid to a culture, 10:03- extract that culture
1:23- J45181 time course starts, 2:23- take out J45181 with salicylic acid added, 5:23- add salycilic acid to a culture, 6:23- extract that culture, 9:23- add salicylic acid to a culture, 10:23- extract that culture, 1:23- add salicylic acid to a culture, 2:23- extract that culture, 5:23- add salicylic acid to a culture, 6:23- extract that culture, 9:23- add salicylic acid to a culture, 10:23- extract that culture