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

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Update

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.

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

10. Try growing up AB3257 again

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