IGEM:MIT/2006/Index: Difference between revisions

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#Quantification
#Quantification
#*Look up GC use
#*Look up GC use
===4. 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) <cite>Dudareva</cite>.
*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 OD<sub>600</sub> 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.
<biblio>
#Dudareva pmid=16442655
</biblio>

Revision as of 07:27, 19 June 2006

Projects/Progress

  1. Preliminary Research
    • Determine toxicity of precursors and products (VV)
      • [[../Notebook/2006-6-16#Toxicity |6-16-2006]]
    • Test effects of minimal media on smell
    • Determine the origin of the bad smell (indole?)
    • Explore biosynthesis of precursors
    • Sequence BAMT, SAMT, BSMT
      • From initial Top10 with [[../Notebook/2006-6-13#pET-28 Primers |T7/TPhiR]] on [[../Notebook/2006-6-16#Sequencing |6-16-2006]]
      • Troubleshoot SAMT with [[../Notebook/2006-6-8#SAMT from A.Majus |designed reverse primer]] on [[../Notebook/2006-6-16#Sequencing |6-16-2006]]
  2. Expression in E. coli
    • Transformation into Top10
    • Transformation into BL21 (DE3)
      • [[../Notebook/2006-6-16#BL21 Tranformation |6-16-2006]]
  3. Applications
    • Heat promoter
  4. Yeast incorporation
  5. Quantification
    • Look up GC use
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