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
4. do test gels of BAMT, SAMT, BSMT pcr products to ensure that pcr was successful and that we have our enzyme DNA
5. do a gel extraction or a pcr cleanup of the enzyme DNA
6. do a restriction enzyme/ligation reaction to put the enzyme DNA into biobrick plasmids (i.e. make biobrick parts)

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 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]

Indole: Making a working stock

According to [2], indole was resuspended at 1M in ethanol. They did a test to determine if ethanol had any ill effects on the E. coli and observed none. Also, according to [2], the reported concentration of indole produced by E. coli in minimal media is 150μM, but Wang et al. found LB cultures to have indole at a concentration of 340μM [3].

Therefore, what we did is the following:

• Make a 1M stock solution of indole (117.15 g/mol).
• Made 10-1mL aliquots of 1M indole;

Note: For testing, we should add indole at two different concentrations: 150 μM and 300 μM. This turns out to be 1.5 μL and 3.0 μL, respectively, of 1M indole for a 10 mL culture.

Indole: the Test

Well...

The pure indole (like a kilogram, at least, of it) smells exactly like an E. coli culture: horrible. After making the 1M stock solution of indole, though, we smelled it and it did not smell bad at all. According to wikipedia, indole at low concentrations smells "flowery". 1M indole smelled rather flowery. And as indicated above, the purported concentration of indole that E. coli produces is max 340 μM.

I (André) added the 1.5 and 3 μL indole to LB. There was no bad E. coli smell. In fact, I would even argue that the 300 μM LB smelled better (but it wasn't exactly a blind trial).

Thus, it is looking as if indole is not the main component of the characterisitc smell of an E. coli culture.

2. Garbe TR, Kobayashi M, and Yukawa H. Indole-inducible proteins in bacteria suggest membrane and oxidant toxicity. Arch Microbiol. 2000 Jan;173(1):78-82. DOI:10.1007/s002030050012 |
3. Wang D, Ding X, and Rather PN. Indole can act as an extracellular signal in Escherichia coli. J Bacteriol. 2001 Jul;183(14):4210-6. DOI:10.1128/JB.183.14.4210-4216.2001 |

check table 2: http://www.pubmedcentral.gov/picrender.fcgi?artid=1243473&blobtype=pdf

enzyme test gels

test 1: BSMT successful, positive control successful

1. used 5 microL loading dye + 5 microL sample dna in 10 test wells
2. used a 1 kb dna ladder in 2 control wells