IGEM:MIT/2006/Notebook/2006-6-12

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T-Shirt

WE NEED A T-SHIRT DESIGN!
Jennyn 13:46, 12 June 2006 (EDT): I'd love to help with this if the team needs.

06/12/06 Repeat (from 6/09/06) Transformation Lab

Sources

  • Note, used 1/2 of designated source
  1. SAMT in PET28 1.6 μg
  2. BAMT in PET28 2.0 μg
  3. BSMT in PET28 2.0 μg

Dehydrated DNA Recovery (from paper)

  • Final DNA Concentration: 1ng/μL (room for better quantification)
  1. Add appropriate amount of TE buffer to 1.7mL Eppendorf tubes (500μL for BAMT and BSMT; 400μL for SAMT).
  2. Carefully cut out paper that contains DNA and place into Eppen.
    • Ensure submergence with toothpick
    • Wait for 10 minutes

Transformation

  1. Followed chemical transformation protocol listed here.
    • Cell Types Transformed:
      • DH5&alpha (big clear tubes with green tape);, Top 10 (Invitrogen) (purple caps), Top 10 (Tom) (big clear tubes), BL21 (Invitrogen) (greenish brown caps)
    • Used 2μL of plasmid/DNA.
    • Heat shocked the BL21(DL3) cells
      • 42 c for 50 seconds
    • Used 500μL 2xYT media for DH5&alpha cells
    • Used 500μL S.O.C. media for other 3 cells
    • horizontal shaking for 60 minutes
    • Plated 200μL cells

Notes

  1. Invitrogen Top 10 BSMT tube fell. It was put on the microfuge for a second to get the cells off the side of the tube.
  2. For TOP10 cells use this transformation protocol, which is similar but slightly different from the what we did previously

Updates on Research in Biosynthetic Pathways for Acid Production

  1. I emailed out the two referenced papers on this pathway: CA-->BA-->SA
  2. Unfortunately, it seems that the gene's sequence encoding the enzyme responsible (benzoic acid 2-hydroxylase) for catalyzing the BA-->SA reaction is not in Pubmed. It seems that the last paper written on the enzyme was in 2000 and did not seem to have any information on sequence information.
  3. Here is a piece from a paper that seems to explan the CA-->BA reaction. "Our results revealed the metabolic pathway that leads from l-phenylalanine to the major aryl metabolites produced by B. adusta. The presence of 13C-labelled trans-cinnamic acid together with PAL activity shows that trans-cinnamic acid is a key pathway intermediate. In addition, trans-cinnamic acid is an efficient precursor of benzoic acid, benzyl alcohol, and benzaldehyde. trans-Cinnamic acid can be subsequently hydroxylated to β-hydroxyphenylpropionic acid (Fig. 2, pathway 2), which in turn can be converted via a β-oxidation step to benzoic acid. This was confirmed by the presence of acetophenone as a degradation product of β-hydroxyphenylpropionic acid. Furthermore, trans-cinnamic acid is the precursor that is most efficiently converted to benzoic acid among the putative precursors which we tested. This confirms that there is a β-oxidation process in B. adusta and that benzoic acid is the major product of this process. β-Oxidation has been found previously in several other fungi (13), although apparently not in B. adusta. To our knowledge, this is the first time that β-oxidation of this nature has been found in a white rot fungus." http://www.pubmedcentral.gov/articlerender.fcgi?artid=92016
  4. http://www.biology.lsa.umich.edu/research/labs/pichersky/references/pub16.pdf
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