IGEM:MIT/2006/Notebook/2006-11-1

Yeast Stuff

1. Yeast strain (ACLY379) finally grew up overnight. Diluted back at 9:45 AM, and will commence transformation after SB lunch.
2. I let the gel of yesterday's digest results run a bit longer to see the exact length of the vectors, and it appears that most of our vectors are just the backbone vector, no insert.
   1. But there are faint bands that might be the correct length. I'll rerun and gel extract, if the bands are visible enough.
3. Transformation
   1. Use high-efficiency protocol because we want to give this transformation the best shot possible.
   2. Transform the amalagam of G5, G6, G7 digests from Oct. 31.

Assemblies

• Have decent number of colonies for all 4 assemblies.
• Barry is screening J45320.J45180. All others are either wrong (due to pSB1AT3-J45400 being wrong) or pointless at this time.

Overnights

• Grew pSB1AT3-J45200 in the wrong media ... Amp-Cm.
• pSB1AC3-J45250/pSB3K3-J45400 didn't grow (either one).
  • But the one started on 30th evening is grown up.
• Neither the pSB1AK3-J45180 big or small colonies grew up in culture (reshma)
  • One of my 45180 colonies (big) did grow, the other (small) didn't (austin)

Streak tests

• pSB1AT3-J45320 grows on Amp-Tet.
• pSB3K3-J45400 grows on Kan.
• pSB1AK3-J45180 grows on Kan.
• pSB1AT3-J45120 grows on Amp-Cm and Amp-Tet.
• pSB1AT3-J45400 grows on Amp-Cm but not Amp-Tet.
  • Agrees with my liquid culture results (austin)
  • Sent out "1AT3-J45400" for sequencing with VF2/VR (austin)
  • Jason clarified. This construct is totally wrong. Reverting back to pSB3K3 version.
• pSB1AC3-J45700 grows on Amp-Cm.
• pSB1AC3-J45250 grows on Amp-Cm.
• I got one colony of pSB1AT3-J45200 on an Amp plate. Maybe slight growth on Tet.

To do

• Make electrically competent indole deficient strain (done)
• Determine the best way to screen colonies. Find primers that can be used to determine if parts are in properly.
  • For construct J45320.J45180, use pchA mut-f and osmY-r
  • For construct J45250.J45400, use ATF1 mut-f and BAT2 mut-r
• Grow up in liquid culture some colonies from each plate during day. (Barry)
• Miniprep + transform indole(-) strain tonight. (cancelled)
• Transform J45250 into IK cells. (Austin and Reshma).
• Ligate and transform J45250.J45400 (from pSB3K3) (Austin and Reshma).
• Grow cultures of (done, Reshma)
  • pSB3K3-J45400
  • pSB1AK3-J45180
  • pSB1AT3-J45200
  • pSB1AC3-J45250
  • pSB1AC3-J45700