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

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.)
 * In the Seo et al., paper, JA is converted to methyl jasmonate outside of a cell by the purified JMT protein product. Thus, we do not know whether jasmonic acid can be taken up by E. coli.

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 in plants.


 * 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

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

Check out this picture for Salicylic Acid Biosynthesis from Phenalaline -- it is much more detailed and is linked into an excellent paper

http://web.mit.edu/boyuan/Public/iGEM/igem6706.gif

Paper Link: http://www.pubmedcentral.gov/articlerender.fcgi?tool=pubmed&pubmedid=11607533

Link (in french): http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T36-447MWH5-4X&_coverDate=11%2F15%2F1971&_alid=412138922&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=4938&_sort=d&view=c&_acct=C000022659&_version=1&_urlVersion=0&_userid=501045&md5=73f9e2c6e28f37feb45c9ef8c9c84ad5

http://www.pubmedcentral.gov/articlerender.fcgi?tool=pubmed&pubmedid=15932991#r10

jasmonic acid

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

BA

 * Immediately following exposure of E. coli to benzoate (20 mM = 2.42&mu;g/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) BA pmid=9393730

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 &mu;g/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
 * phBSMT1- None. phBSMT2- EcoRI site found in the sequence at base pair 1140.
 * 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
 * BSMT- Ported to "E. coli"
 * Registry accounts
 * All- 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)
 * or we can just get the DNA from yeast


 * BL21(DE3)+pLysS competent cells (the Endy Lab has these)
 * Arab. JMT
 * primers: http://openwetware.org/wiki/IGEM:MIT/2006/Notebook/2006-6-8

Parts in the registry
BBa_J45000 BBa_J45001 BBa_J45002 BBa_J45003 BBa_J45004 BBa_J45005 BBa_J45006 BBa_J45007 BBa_J45013