IGEM:MIT/2006/Notebook/2006-6-7

Compound Database

Transport

• SA can be transported into/out of tobacco cells (http://ejournal.sinica.edu.tw/bbas/content/1999/4/bot404-03.html)
• BA was added exogenously, and methyl benzoate (MB) was produced, so BA can enter E. coli. MB concentrations were measured without needing to lyse the cells, so MB can exit the cell. (Reference; it is the first paper under this section)
• In an experiment done by Horton et al., isoamyl alcohol was added exogenously and was taken up by the E. coli. Concentration of the product, isoamyl acetate, was then measured without lysing the cells, so we know that the product will be excreted. (see Horton et al. paper in this section.)
• The only sentence in which Seo et al., discusses JMT usage in E. coli is in the following sentence:

"Recombinant JMT protein in Escherichia coli catalyzed the formation of methyl jasmonate from jasmonic acid with Km value of 38.5 µM."

When discussing transport in plants, JA is a hormone that is freely flowing through the organisms. Thus, JA is added to the cells exogenously in the naturally-occurring mechanism.

• E. coli cells expressing C. breweri BEAT produced enzymatically active protein, and also synthesized benzylacetate and secreted it into the medium
  • http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=9628024&dopt=Abstract

Biosynthesis

salicylic acid/benzoic acid

http://www.hort.purdue.edu/rhodcv/hort640c/secprod/sec7.gif

jasmonic acid

• Many pathways described at http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1066929&blobtype=pdf

Toxicity

BA

• Immediately following exposure of E. coli to benzoate (20 mM = 2.42μg/mL), the cell doubling rate reduced 10-fold (this is at pH 6.5; at pH 8.0, there is no significant change in doubling rate). But we have to keep in mind that benzoate was not being metabolized, so benzoate was simply accumulating in the cell, affecting the pH (this is what was being studied, actually). Methyl benzoate, since it is uncharged, may not have this same effect.

1. Lambert LA, Abshire K, Blankenhorn D, and Slonczewski JL. Proteins induced in Escherichia coli by benzoic acid. J Bacteriol. 1997 Dec;179(23):7595-9. DOI:10.1128/jb.179.23.7595-7599.1997 |

Concentration

What ranges of concentrations should we be adding?

banana

• 10 mM isoamyl alcohol

jasmine

• 1 mM JA and 1 mM S-adenosyl methionine (SAM)

salicylic acid or benzoic acid

• SAMT (A. Majus and C. breweri) 5 μg/ml of SA (or BA)

Sequence analysis

• Restriction sites
  • JAMT- None.
  • SAMT (A. Majus and C. Breweri) - None
  • ATF1- EcoRI site found @ base pair 412
• Codon optimization
  • JAMT- Already transferred to E. coli and S. cerevisiae
  • SAMT - (A. Majus and C. Breweri) - Already ported to e. coli
  • ATF1- Already ported to E. coli
• Registry accounts
  • Stephen- Yes
  • Kate- Yes
• Install DNA 2.0 software?
  • Stephen- Yes
  • Kate - Yes (although have not used)

Things to get

• Precursor molecules etc.
• To make isoamyl acetate:
  • isoamyl alcohol
  • contact Horton et al. (contact Frederick B. Rudolph. e-mail: fbr@rice.edu ; phone: (713) 348-4014)

• BL21(DE3)+pLysS competent cells (the Endy Lab has these)
• Arab. JMT

Parts in the registry

<bbpart>BBa_J45000</bbpart> <bbpart>BBa_J45001</bbpart> <bbpart>BBa_J45002</bbpart> <bbpart>BBa_J45003</bbpart> <bbpart>BBa_J45004</bbpart> <bbpart>BBa_J45005</bbpart> <bbpart>BBa_J45006</bbpart> <bbpart>BBa_J45007</bbpart> <bbpart>BBa_J45011</bbpart>