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From 2010.igem.org

July 8

The phsABC plasmid worked! We have metal precipitation! Woooo!

1. Cloning issues

• Did the double digestion of the terminator work?
  • The EcoRI was old and might have failed, check each enzyme on a gel
• SpeI not very efficient in EcoRI buffer, will try to incubate longer
• The ligation didn't work, but there are lots of ways to keep trying. This is a great way for people to come help during the week.

2. Copper precipitation assay

• Pick a concentration from the cell growth assay, like 1 mM Cu?
• Grow shaking, at time points spin out the cells and measure the absorbance
  • this is assaying for loss of copper

3. Does phsABC confer copper resistance?

• 96-well plate assay
• same as before, but with the phsABC plasmid
  • Since Leah has this assay dialed-in, this is a great job for a part-time helper to run and analyze!

4. Cu resistance

• will be shipping in a day or two
  • pRJ1004 (Cu efflux protein) (Jon will email to follow up)
    • massive plasmid, no antibiotic resistance
  • Get together next week to request several plasmids (Tuesday morning at 10 am).

5. Email Kara about the mold (Jon)

6. Ways to get students involved (Dan email the group)

• Ask when they're available, delegate a task
• make sure someone is there to help them

July 15