IGEM:MIT/2006/Notebook/2007-6-28

General Update

1. Talked with Professor Tribe of the University of Melbourne yesterday who was extraordinarily helpful. I am still in communication with him about how to best overproduce chorismate. For now, I will ask whether Yale's center has two types of strains suggested. Strain AB3249 in this paper should work: http://jb.asm.org/cgi/reprint/93/1/237?view=long&pmid=5335890

2. HPLC- Let's hope this works

3. Time courses looked decent, will modify the following:

• 1. Will dilute a 5-mL grown culture (15 uL in 150 mL) for time courses from now on (will do this from LC of a masterplate colony). Realized that before I took directly from the glycerol, which could have given rise to variations in growth between J45120 and J45181.

• 2. Will take OD600 readings after GC sample extraction to reduce evaporation

• 3. Will now move to two-hour time intervals to gain more data points.

• 4. Will only go up to the 13-hour mark since cells seem to die after that or at least stopped producing significant amounts of methyl salicylate in both J45120 and J45181 cultures.

• 5. So, the procedure would go as follows:

0:00- J45120 time course starts, add salicylic acid to a culture 1:00- extract that culture, 2:00- add salycilic acid to a culture, 3:00- extract that culture, 4:00- add salicylic acid to a culture, 5:00- extract that culture, 6:00- add salicylic acid to a culture, 7:00- extract that culture, 8:00- add salicylic acid to a culture, 9:00- extract that culture, 10:00- add salicylic acid to a culture, 11:00- extract that culture, *if necessary* 12:00- add salicylic acid to a culture, *if necessary* 13:00- extract that culture

0:20- J45181 time course starts, add salicylic acid to a culture 1:20- extract that culture, 2:20- add salycilic acid to a culture, 3:20- extract that culture, 4:20- add salicylic acid to a culture, 5:20- extract that culture, 6:20- add salicylic acid to a culture, 7:20- extract that culture, 8:20- add salicylic acid to a culture, 9:20- extract that culture, 10:20- add salicylic acid to a culture, 11:20- extract that culture, *if necessary* 12:20- add salicylic acid to a culture, *if necessary* 13:20- extract that culture

Things to Do Today

1. Add 15 uL grown 5-mL J45181 culture from last night to 150-mL LB AMP/TET solution (250-mL flasks) twice. Grow up for 20 mins at 220 RPM- DONE

2. Split the two cultures into 12 25-mL cultures in 12 250-mL flasks and grow up at 110 RPM- DONE

3. For every hour excluding the first hour for 12 hours (hours 2-13: 12:19 OD600=0.00, 1:19, 2:19, 3:19, 4:19, 5:19, 6:19, 7:19, 8:19, 9:19, 10:19, 11:19) take OD readings to construct growth curve as Francois suggested- DOING (STOPPED AT 1:19!!! Drew said to make a master plate & use the same liquid culture to redo these experiments)

4. Make new solution of water and isoamyl acetate (8.8 uL added per 20 mL water)- DONE

5. Run new solution on HPLC- DONE (DID NOT WORK, Andreas said we can try it under different conditions tomorrow at a neutral pH, he thinks that at the low pH we were running it at, the compound falls apart)

6. Write back Professor Tribe to run by plan and confirm I heard him right on the phone- DONE

7. Contact Yale center about strains Tribe suggested- DONE

8. Submit sequencing of J45320 to see if there is the same mutation present on J45800- DONE

9. Organize lab bench area- DONE

10. Make master plates of J45120, J45181, J45700 (A), J45800, J45200, J45250, J45600, J45900 (A), J45995, J45996, R0040.E0840, B0015- DONE

11. Research leucine pathway

12. Get rid of 25-mL cultures made at beginning of day- DONE

Leucine Pathway Research

Interesting: This is a big reason why J45600 and J45900 did not smell like banana when supplemented with L-leucine

"Leucine transport activity is repressed by growing the cells with L-leucine supplementation. According to data given by Wood, the total uptake activity of a repressed cell (cells grown in the presence of leucine (0.05 g/L) is reduced to half of the activity of unrepressed cell (cells grown in minimal media without leucine)"- http://www3.interscience.wiley.com/cgi-bin/fulltext/107622894/PDFSTART. Our cells were grown with ~2 g/L leucine added to the media.

Pathway

http://biocyc.org/NEW-IMAGE?type=PATHWAY&object=BRANCHED-CHAIN-AA-SYN-PWY- looks like tyrB and ilvE are important

Amount of leucine required for 40 mins of cell growth: 428 umol/g

http://www3.interscience.wiley.com/cgi-bin/fulltext/71004055/PDFSTART

Assuming 1 cell is 3*10^-13 g http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi and at exponential phase (as assumed in the paper above):

OD600~1.3---> ~10^9 cells/mL (http://openwetware.org/wiki/BE.109:Systems_engineering/Device_characterization)---> ~10^10 cells/10 mL---> 10^10*3*10^-13 g = 3 mg cells/10 mL----> ~1.1 umol leucine in 10 mL ---> ~.11 mM

In 10 mL culture, we would add 4.4 uL isoamyl alcohol (or .44 mg/L or ~4 mM)---> assuming 100% efficiency ---> want ~4 mM leucine in cell