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

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#Species: Arabidopsis thaliana (thale cress)
#Species: Arabidopsis thaliana (thale cress)
#Sequence & Structure
#Sequence & Structure
#*The gene JMT was isolated from a cDNA (22) and a genomic library of A. thaliana (Columbia ecotype) by screening with the Brassica NTR1 (20). The genomic library was constructed into the XhoI site of partially filled λFixII vector (Stratagene, Heidelberg, Germany). Inserts in the phage DNAs hybridizing with the NTR1 probe were isolated and subcloned into pBluscriptSK(+)II vector. Nucleotide sequences of the overlapping partial clones were determined by using an automated DNA sequencer (Applied Biosystems, model PRISM 377).
#*Use cDNA from A. thaliana (multistep- screened with Brassica NTR1)
#*JMT cDNA was inserted into the pGEX-2T vector (Amersham Pharmacia) at the EcoRI site and fused with glutathione-S-transferase gene (GST). The recombinant was transformed into E. coli BL21.  
#*JMT cDNA inserted into the pGEX-2T vector (Amersham Pharmacia) at the EcoRI site and fused with glutathione-S-transferase gene (GST).  
#*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?
#**Use cDNA from arabadopsis
#Reaction catalyzed
#Reaction catalyzed
#*Substrate
#*Substrate

Revision as of 12:46, 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) 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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