IGEM:MIT/2006/Notebook/2006-6-19

To Do

1. troubleshoot bad atf1 PCR with advisors and possibly repeat with exhaustive temperature controls
2. smell methyl salicylate, methyl benzoate, indole (if order arrives) -- how much does it take to make a culture smell??
3. hopefully do tests with minimal media/plating/etc using the BL21 transformants from overnight weekend cultures

Repeat ATF1 PCR

1. 8 sample tubes, 4 controls
   • (-): no template, (-): no primer, (+): same template, (+): new primer template
   • Sample master mix 9x, control master mix 5x, [[../2006-6-15 | from here]]
   • Temperature gradient from 40-50

Yeast Incorporation

Farhi et al. (including Dudareva) have been able to express the A. majus (snapdragon) BAMT in S. cerevisiae and produce methyl benzoate. The following are what I think were major procedural points and general conclusions that came out of the work. Just to note, all of this information, unless otherwise noted, came from this reference (the one below) [1].

• Procedural Points
  • They used a citrate buffered minimal medium (pH 4.5 or 6) for BA treatment. (This was presumably NOT in order to achieve a better smelling culture.
  • The volme they used for GC analysis was generally 10mL (I'm pretty sure everyone figured out that it really wasn't 25L; I was mistaken, obviously).
  • They grew the culture overnight to OD₆₀₀ of 0.5, added the BA and other necessary inducing agents, and checked the methyl benzoate production after 6 hours of growth.
• General Points/Conclusions
  • "BA is a known retardant of yeast growth at concentrations above 2mM..."
  • Methyl benzoate production was relatively linear over the first 24 hours after substrate addition (without leveling).
  • They used two different BAMT "inducing systems": constitutive expression from the TPI promoter and a Cu-inducible expression system. The inducible expression system led to at least one order of magnitude greater methyl benzoate production.
  • They showed that 85% of the methyl benzoate produced is able to escape into the culture medium.
  • When speculating on the relatively low efficiency of their methyl benzoate production system (~1%), they noted that Pdr12 is a yeast membrane pump that has been reported to pump benzoic acid out of the cell.

1. Farhi M, Dudareva N, Masci T, Weiss D, Vainstein A, and Abeliovich H. Synthesis of the food flavoring methyl benzoate by genetically engineered Saccharomyces cerevisiae. J Biotechnol. 2006 Apr 10;122(3):307-15. DOI:10.1016/j.jbiotec.2005.12.007 |

GC/MS

1. Contacted Janusz Pawliszyn about GC/MS use with methyl benzoate
2. Yea! We have the documentation on the appropriate instrumentation and procedure for GC with methyl benzoate [J_Sep_Sci_2005_mold.pdf here]

New SAMT Sequencing primer (on hold)

Choose and order a new sequencing primer forward on the SAMT construct. We hope to sequence into the region where sequencing failed at the transition from pET28a backbone to the insert at the BamHI site.

Smell Test of new transformants

Control	BAMT/BA	SAMT/SA	BSMT/BA	BSMT/SA
10mL LB + 20μL BL21 cells + 8.2μL BA [in a tube]	10mL LB Kan + 20μL BAMT cells + 8.2μL BA [in a tube]	10mL LB Kan + 20μL SAMT cells + 6.2μL SA [in a tube]	10mL LB Kan + 20μL BSMT cells + 8.2μL BA [in a tube]	10mL LB Kan + 20μL BSMT cells + 6.2μL SA [in a tube]
10mL minimal media + 20μL BL21 cells + 8.2μL BA [in a tube]	10mL minimal media Kan + 20μL BAMT cells + 8.2μL BA [in a tube]	10mL minimal media Kan + 20μL SAMT cells + 6.2μL SA [in a tube]	10mL minimal media Kan + 20μL BSMT cells + 8.2μL BA [in a tube]	10mL minimal media Kan + 20μL BSMT cells + 6.2μL SA [in a tube]