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.

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 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.