IGEM:MIT/2006/Notebook/2007-7-9

General Update

1. Heard back from Dr. Esaki last night. He is sending us the leucine-overproducing plasmid.

2. Supposed to meet with Drew this afternoon.

3. Waiting to hear from geobiology to see if isoamyl acetate was detected by the GC (was not detected but Alex suspects the settings just need tweeked).

4. Professor Calvo said that he had a pathogen Salmonella strain which overproduces leucine.

5. ATCC has nothing for us in regard to an overproducing leucine strain.

6. John Wertz from Yale gave us some alternative options on how to overproduce leucine in E. coli

Things to Do

1. Start a J45181 time course in which cultures' OD600 are taken every hour for hours 3-14 of growth. The initial cultures are in 2 250-mL flasks containing 150 mL of media and 15 uL of stock LC. Then, the culture is split into 12 250-mL flasks. After talking with Jason, I decided to just take three of these baffled flasks to construct a growth curve, removing 500 uL at each time point and averaging them- WORKING ON IT

2. Look up prices of baffled 250-mL flasks online- DONE

3. Ask Isadora about ordering more baffled 250-mL flasks- DONE (6 more on the way)

4. Ask Isadora about ordering more heptane- DONE (2 1-L containers on the way)

5. See if others in the lab can smell the preserved large cultures of J45700 and J45900- DONE (Jason could smell both, Barry maybe smelled banana, Nishant could smell mint)

6. Respond to Professor Calvo about Salmonella strain. Consult with Drew first

7. Write/edit abstract and title for microbial genetics conference- DONE

8. Send abstract to Veena before sending to Drew- DONE

9. Send abstract to Drew- DONE

10. Have lunch with Veena to update her on the project- DONE

11. Send ordering info for more cuvettes to Isadora- DONE (on the way)

12. Respond to Wertz about over-producing leucine strain- DONE (told him we'd order the ilvE- strain)

13. Make new LCs of J45120 and J45181- DONE

14. Develop a protocol for time course measurements- DONE

15. Ask Alex about second runs on the GC- DONE

16. Ask Dr. Esaki if plasmid makes all of the leucine from cell's natural stock of alpha-ketoisocapriote- DONE

17. Set up experiment Drew suggested (2 flasks of each J45600, J45700, J45800, & J45900, one with precursor added, one without)- DONE

Topics for Meeting with Drew

1. Update on chorismate-overproducing strain- obtaining aroF- aroG- aroH- and introducing two mutations to the chromosome

2. Update on leucine-overproducing strain- Japanese group sending us a plasmid known to overproduce leucine in E. coli, Alternative options- obtain IlvE- strain from Yale and A) PCRing tyrB and putting on a high-copy plasmid or B) obtain a tyrB plasmid from Japan (OUT, takes 1 month after signing a MTA), last option- Salmonella

3. Time Course Experiments

4. GC and isoamyl acetate

5. Priorities- What is more important: characterizing what we have or improving composite devices?

Protocol for Time Course

Hour 4- add SA

Hour 5- add SA, GC extract

Hour 6- add SA, GC extract

Hour 7- add SA, GC extract

Hour 8- add SA, GC extract

Hour 9- add SA, GC extract

Hour 10- add SA, GC extract

Hour 11- add SA, GC extract

Hour 12- add SA, GC extract

Hour 13- GC extract

At each hr, remove both culture with added SA and culture with SA to be added. Quickly put culture with added SA in fridge, while you add SA and clean the separatory funnel

Growth Curve Construction

• Note all OD600s were extrapolated from 1:2 dilutions of the cultures

Hour--------------Culture 1------------Culture 2---------Culture 3--------Average

3-----------------0.02------------------0.04---------------0.02-------------0.027 (lag)

4-----------------0.08------------------0.08---------------0.08-------------0.080 (lag)

5-----------------0.28------------------0.24---------------0.26-------------0.260 (lag)

6-----------------0.74------------------0.70---------------0.70-------------0.713 (lag/exp)

7-----------------1.48------------------1.40---------------1.38-------------1.420 (exp)

8-----------------2.32------------------2.20---------------2.30-------------2.273 (exp)

9-----------------2.68------------------2.56---------------2.68-------------2.640 (exp/sta)

10---------------2.84-------------------2.68---------------2.84-------------2.787 (sta)

11---------------2.90-------------------2.74---------------2.88-------------2.840 (sta)

12---------------2.92-------------------2.80---------------2.86-------------2.860 (sta)

13---------------2.94-------------------2.80---------------2.88-------------2.873 (sta)