IGEM:MIT/2006/Notebook/2006-6-7: Difference between revisions

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*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.  ([[IGEM:MIT/2006/Notebook/2006-6-6#BAMT: SAM-Benzoic Acid Carboxyl Methyltransferase|Reference]]; it is the first paper under this section)
*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.  ([[IGEM:MIT/2006/Notebook/2006-6-6#BAMT: SAM-Benzoic Acid Carboxyl Methyltransferase|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 [[IGEM:MIT/2006/Notebook/2006-6-6#Alcohol_Acetyltransferase.28s.29:_ATF1_.26_ATF2|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 [[IGEM:MIT/2006/Notebook/2006-6-6#Alcohol_Acetyltransferase.28s.29:_ATF1_.26_ATF2|this section]].)
*The only sentence in which Seo et al., discusses JMT usage in ''E. coli'' is in the following sentence:
*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.


"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 in plants.
 
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
*E. coli cells expressing C. breweri BEAT produced enzymatically active protein, and also synthesized benzylacetate and secreted it into the medium
Line 16: Line 14:
====salicylic acid/benzoic acid====
====salicylic acid/benzoic acid====
http://www.hort.purdue.edu/rhodcv/hort640c/secprod/sec7.gif
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====
====jasmonic acid====
*Many pathways described at http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1066929&blobtype=pdf
*Many pathways described at http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1066929&blobtype=pdf
===Toxicity===
===Toxicity===
====BA====
====BA====
Line 41: Line 52:
**SAMT (A. Majus and C. Breweri) - None
**SAMT (A. Majus and C. Breweri) - None
**ATF1- EcoRI site found @ base pair 412
**ATF1- EcoRI site found @ base pair 412
**phBSMT1- None. phBSMT2- EcoRI site found in the sequence at base pair 1140.
*Codon optimization
*Codon optimization
**JAMT- Already transferred to ''E. coli'' and ''S. cerevisiae''  
**JAMT- Already transferred to ''E. coli'' and ''S. cerevisiae''  
**SAMT - (A. Majus and C. Breweri) - Already ported to e. coli
**SAMT - (A. Majus and C. Breweri) - Already ported to e. coli
**ATF1- Already ported to ''E. coli''
**ATF1- Already ported to ''E. coli''
**BSMT- Ported to "E. coli"
*Registry accounts
*Registry accounts
**Stephen- Yes
**All- Yes
**Kate- Yes
*Install DNA 2.0 software?
*Install DNA 2.0 software?
**Stephen- Yes
**Stephen- Yes
Line 59: Line 71:
**isoamyl alcohol
**isoamyl alcohol
**contact Horton et al. (contact Frederick B. Rudolph. e-mail: fbr@rice.edu ; phone: (713) 348-4014)
**contact Horton et al. (contact Frederick B. Rudolph. e-mail: fbr@rice.edu ; phone: (713) 348-4014)
**or we can just get it from yeast
**or we can just get the DNA from yeast


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


==Parts in the registry==
==Parts in the registry==
Line 73: Line 86:
<bbpart>BBa_J45006</bbpart>
<bbpart>BBa_J45006</bbpart>
<bbpart>BBa_J45007</bbpart>
<bbpart>BBa_J45007</bbpart>
<bbpart>BBa_J45011</bbpart>
<bbpart>BBa_J45013</bbpart>

Latest revision as of 14:37, 9 June 2006

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.)
  • 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.

Biosynthesis

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

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 | PubMed ID:9393730 | HubMed [BA]

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
    • 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)

Systems & Applications

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

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_J45013</bbpart>

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