IGEM:MIT/2006/Notebook/2007-7-16

=Update=



https://catalog.invitrogen.com/productImages/1900/1896.GIF

1. Ran a protein gel over the weekend using the protocols http://openwetware.org/wiki/Knight:Protein_solubility and http://openwetware.org/wiki/Knight:NuPAGE_electrophoresis/Hybrid_staining with modifications step 6 of protein solubility soluble and insoluble fractions: centrifuge lysate at 10000 g for 30 mins at 4 degrees C

2. The gels did not look good. BAT2 looked pretty good. THI3 looked absent. The pchB band looked present in both the control and experimental lanes. The pCHA band looked absent. Addditionally, BSMT looked absent from all of the lanes (each lane of gel 1 contained a device with the BSMT generator). ATF1 may have been present in all of the lanes (each lane of gel 2 contained a device with the ATF1 generator). We will discuss these results at the meeting today.


 * BAT2 = 41.63 kilodaltons
 * THI3 = 68.38 kilodaltons
 * ATF1 = 60.98 kilodaltons
 * PchB = 11.452 kilodaltons
 * PchA = 52.290 kilodaltons
 * BSMT1 = 40.769 kilodaltons



=What to Do=

1. Compile efficiencies of all of the enzymes.

For pCHA- kcat=43.1 1/min, Km=4.5 uM, kcat/Km = 1.6 * 10^5 1/M*s http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=12624097&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

For pCHB- kcat= 1.06 1/s, Km=.95 uM, kcat/Km = 1.12 *10^6 1/M*s http://www.jbc.org/cgi/content/full/280/38/32827

Also does chorismate-->prephenate, kcat=.085 1/s, Km=38 uM, kcat/Km=2.24*10^3

For BSMT- kcat/Km= 230 1/M*s

2. Check to see if all of the enzymes were ported to E. coli successfully.

pchBA fusion protein originally from Pseudomonas aeruginosa expressed in Arabidopsis thaliana.

BAT2 and THI3 have not been expressed in organisms other than S. cerevisiae as far as I know.

3. Make a reservation for a room in 68- DONE (68-674)

4. Let everyone know about the meeting today- DONE

5. Prep for the meeting- DONE

6. Prepare isoamyl acetate samples for calibration curve with internal and external standards (they came in today)- DONE

Mix- 100 ppm n-hexyl acetate, 100 ppm octyl acetate, 5, 10, 25, 100, 250, and 500 ppm isoamyl acetate, in heptane

7. Submit isoamyl acetate samples to Alex- DONE

8. Construct growth curves for J45200 and J45250 from data collected Friday- DONE

9. Make new LCs of J45120/J45181- DONE

10. Try growing up AB3257 again- DONE

=Long-term Plan=

Tasks:

1. Check sequencing of J45600 and J45800 (tomorrow)

2. Check RNA secondary structures of precursor devices (look at it with Barry tomorrow)

3. Exponential phase protein gel (througout the week)

4. Fluorescent experiment (ask Barry when the plate reader is available)

5. Redesign device characterization experiment (tonight)

6. Media (ask Knight about this, get ideas about leucine pathway)

=Tentative Schedule=

Tonight- Grow up cells to exponential phase for protein gel tomorrow, develop new protocol for time course, ask Barry about when the plate reader is available, ask Knight about media

Tomorrow- Develop growth curves for J45120 and J45181 with new protocol, run protein gel

Wednesday- Run time course for J45120 and J45181 with new protocol, destain and view protein gel

Thursday- Run GC samples

Friday- Tahoe

=New Protocol for Time Course Experiment=

1. Add 25 uL of grown J45181 and J45120 cultures to 250 mL of media in 1-L flask.

2. Grow culture up at 220 RPM for 20 mins.

3. Grow culture up at 110 RPM (this is time zero).

4. At n hrs of growth, extract 25-mL culture and place into 125-mL flask, add SA

5. At n+1 hrs of growth, GC extract culture from 125-mL flask

6. Repeat steps 4-5 for n=4 hrs to n=12 hrs in 1-hr increments

7. In order to do J45181 and J45120 cultures in parallel, stagger J45181 and J45120 by 20 mins.