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

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#Sequence & Structure
#Sequence & Structure
#*Use cDNA from A. thaliana (multistep- screened with Brassica NTR1)
#*Use cDNA from A. thaliana (multistep- screened with Brassica NTR1)
#*JMT cDNA inserted into the pGEX-2T vector (Amersham Pharmacia) at the EcoRI site and fused with glutathione-S-transferase gene (GST).  
#*JMT cDNA inserted into the pGEX-2T vector (Amersham Pharmacia-GE) at the EcoRI site and fused with glutathione-S-transferase gene (GST).  
#*The recombinant was transformed into E. coli BL21. Fusion protein GST-JMT was expressed
#*The recombinant was transformed into E. coli BL21. Fusion protein GST-JMT was expressed
#**Purified to get JMT- issue?
#**Purified to get JMT- issue?
#Reaction catalyzed
#Reaction catalyzed
#*Substrate
#*Substrate: Jasmonic acid and SAM added exogenously
#**Jasmonic acid and SAM added exogenously
#*Product: Methyl jasmonate
#*Product: Methyl jasmonate
#**Given off by plants as defense, also in jasmin oil
#**Given off by plants as defense, also in jasmin oil

Revision as of 12:48, 6 June 2006

A very brief description of the mechanism for making cinnamon smells:

http://docs.lib.purdue.edu/dissertations/AAI9939353/

We'll dump all this information on today's page for now but you guys should think about what page structure you want to use to store all your information!

Enzyme 1

  1. Species
  2. Sequence & Structure
    • Codon optimized?
    • Restriction sites
    • Post-translational modification
    • Material availability
      • cDNA from arabadopsis
      • Request from authors
        • Form of DNA?
      • need to synthesize?
  3. Reaction catalyzed
    • Substrate
      • Synthesized in our chassis or supplied exogenously?
    • Product
      • Coolness (Austin)
    • Reaction rates - Km,kcat
  4. Does it smell??
    • Regulation issues
    • Transport issues for substrate/product
  5. Ported to E. coli or yeast?
  6. What is the regulation in planta
  7. References & Links
    • Corresponding authors
    • Genbank accession numbers
    • Pubmed
    • Websites
    • Any companies or patents?


Kate's enzyme

  1. Species
  2. Sequence & Structure
    • Codon optimized?
    • Restriction sites
    • Post-translational modification
    • Material availability
      • cDNA from arabadopsis
      • Request from authors
        • Form of DNA?
      • need to synthesize?
  3. Reaction catalyzed
    • Substrate
      • Synthesized in our chassis or supplied exogenously?
    • Product
      • Coolness (Austin)
    • Reaction rates - Km,kcat
  4. Does it smell??
    • Regulation issues
    • Transport issues for substrate/product
  5. Ported to E. coli or yeast?
  6. What is the regulation in planta
  7. References & Links
    • Corresponding authors
    • Genbank accession numbers
    • Pubmed
    • Websites
    • Any companies or patents?

Jasmonic acid carboxyl methyltransferase

  1. Species: Arabidopsis thaliana (thale cress)
  2. Sequence & Structure
    • Use cDNA from A. thaliana (multistep- screened with Brassica NTR1)
    • JMT cDNA inserted into the pGEX-2T vector (Amersham Pharmacia-GE) at the EcoRI site and fused with glutathione-S-transferase gene (GST).
    • The recombinant was transformed into E. coli BL21. Fusion protein GST-JMT was expressed
      • Purified to get JMT- issue?
  3. Reaction catalyzed
    • Substrate: Jasmonic acid and SAM added exogenously
    • Product: Methyl jasmonate
      • Given off by plants as defense, also in jasmin oil
    • Reaction rates - Km: 38.5 µM; kcat
  4. Does it smell??
    • Methyl jasmonate has jasmin smell
    • Regulation issues
    • Transport issues for substrate/product
  5. Ported to E. coli
  6. Regulated in planta:
  7. References & Links
    • Corresponding authors
    • Genbank accession numbers: BD441046, BD441047

PAL & SAM:CCMT

  1. Species: PAL (Phenylalanine Ammonia-lyase) is found in several organisms, but for our interests, it seems the best option is the gene from Rhodosporidium toruloides. SAM:CCMT (S-adenosyl-L-methionine:cinnamate carboxyl methyltransferase) can only be found in Ocimum basilicum L., Lamieaceae (a basil plant) as far as I can tell.
  2. Sequence & Structure
    • Codon optimized?
    • Restriction sites
    • Post-translational modification
    • Material availability
      • cDNA from arabadopsis
      • Request from authors
        • Form of DNA?
      • need to synthesize?
  3. Reaction catalyzed
    • Substrate
      • Synthesized in our chassis or supplied exogenously?
    • Product
      • Coolness (Austin)
    • Reaction rates - Km,kcat
  4. Does it smell??
    • Regulation issues
    • Transport issues for substrate/product
  5. Ported to E. coli or yeast?
  6. What is the regulation in planta
  7. References & Links
    • Corresponding authors
    • Genbank accession numbers
    • Pubmed
    • Websites
    • Any companies or patents?
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