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

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.

Dehydrated DNA Recovery (from paper)

 * Final DNA Concentration: 1ng/&mu;L (room for better quantification)


 * 1) Add appropriate amount of TE buffer to 1.7mL Eppendorf tubes (500&mu;L for BAMT and BSMT; 400&mu;L for SAMT).
 * 2) Carefully cut out paper that contains DNA and place into Eppen.
 * 3) *Ensure submergence with toothpick
 * 4) *Wait for 10 minutes

Transformation

 * 1) Followed chemical transformation protocol listed here.
 * 2) *Cell Types Transformed:
 * 3) **DH5&alpha (big clear tubes with green tape);, Top 10 (Invitrogen) (purple caps), Top 10 (Tom) (big clear tubes), BL21 (Invitrogen) (greenish brown caps)
 * 4) *Used 2&mu;L of plasmid/DNA.
 * 5) *Heat shocked the BL21(DL3) cells
 * 6) **42 c for 50 seconds
 * 7) *Used 500&mu;L 2xYT media for DH5&alpha cells
 * 8) *Used 500&mu;L S.O.C. media for other 3 cells
 * 9) *horizontal shaking for 60 minutes
 * 10) *Plated 200&mu;L cells

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