IGEM:MIT/2006/Notebook/2007-8-21

=Things to Do=

1. Early morning- run GC samples from last night- DONE (only very small traces of MS found in some samples but hardly any indole found either: cells must not have been spun down enough, also protease inhibitor coelutes at MS time point)

2. Prepare for meeting with Reshma- DONE

3. Discuss with Barry and Reshma plan- DONE (additions to plan: do 15-, 16-, and 17-hour time points adding salicylic acid at the beginning for J45120 and J45181- need to grow up 3 LCs first, streak out fresh plates, do 3 of 15- hour for J45120 and J45181, reconfigure GFP data to synthesis rates)

4. Ask Andreas if he has another protease inhibitor- DONE (after talking with him, we decided there is no need for a protease inhibitor if we know that our protein folds correctly and we keep the cells cold)

5. Ask Isadora about travel arrangements- DONE

6. Make up fresh Tris Buffer- DONE

7. At the end of the day, make three LCs from three different colonies each of J45995, J45996, R0040.E0840, and B0015- DONE

8. At the end of the day, set up J45120/J45181 time course (Time Zero = 7:55/8:15)- DONE


 * 10 uL culture


 * 100 mL media


 * 2 mL salicylic acid

Then split into 3 25-mL cultures

9. At the end of the day, set up banana cultures- DONE:

Make the following cultures to prepare for GC tomorrow:


 * 25-mL J45200 w/ precursor (13.6 uL)


 * 25-mL J45250 (1AT3) w/ precursor (13.6 uL)


 * 25-mL J45200 w/o precursor (control)


 * 80-mL J45600


 * 80-mL J45900

Calculation of Precursor

We initially forgot to take into account isoamyl alcohol's density when deciding at which concentration to add isoamyl alcohol.

We used 4.4 uL/10 mL because we assumed density to be 1 g/mL... Thus, .0044 g/.01 L*1 mol/88.2 g = 5 mM

To make the final concentration be 5 mM, we actually want 4.4 uL/.809 (g/mL density)= 5.44 uL.

10. Reconfigure GFP data to synthesis rates- DONE (looks pretty good)

11. Streak out fresh plates of J45120 and J45181- DONE

12. Make fresh LCs of J45200 and J45250- DONE

=Protocol=

OD600s: Missed 13 hours; J45120 14 hours- 2.48, J45181 14 hours- 2.46; J45120 15 hours- 2.58, J45181 15 hours- 2.50

1. At each time point, aliquot 20 mL cultures (J45120 and J45181) into falcon tubes and remove .5 mL to take an OD600 measurement.

2. Incubate culture for 10 minutes on ice.

3. Centrifuge the samples at 4C for 20 mins at 3800 RPM.

4. Discard the supernatants. Store the pellets at -80C until the last time point.

5. Once all time points are collected, resuspend pellets in 10 mL Tris buffer (10 mM Tris, 100 mM NaCl, pH 8).

6. Centrifuge the samples at 4C for 20 mins at 3800 RPM.

7. Discard supernatants.

8. Resuspend cells in 1 mL B-PER II.

9. Add 19 mL PBS to samples.

10. Add 20 uL 1 Unit/1 uL Benzonase to samples

11. Incubate at 4C for 30 mins

12. Add .4 mL .1 M salicylic acid to samples at staggering 20 min time points.

13. Let sample incubate for 2 hours at room temperature after addition of salicylic acid.

14. GC extract the samples.