Team:UCLA/Notebook/Recombinant Expression/19 May 2015

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• Starter culture failure
  • Yesterday's attempt at creating a starter culture from the 5th plating of the old transformatants did NOT grow. We hit an OD600 of 0.004, basically negligible growth.
    • Created new 11mL LB supplemented with 11uL 1000x Ampicillin (final 1x concentration 50ug/uL) starter cultures with colonies picked from 1/21 platings -- Plates 1,2,4,5.
      • Incubated plates at 37 degrees shaking at 1415. Will check OD of starter cultures at 1900.

• 1L Expression of Tamura construct
  • Inoculated 950 mL of LB supplemented with 1mL of 100x Carbenicillin (final concentration 100ug/uL) with 10mL starter culture grown overnight.
    • Shook cells in 37 degrees Celsius until an OD600 of 0.6 is reached.
    • Induced protein expression using 5mL of 100mM IPTG stock (final concentration 0.5mM).

• Gene expression monitoring
  • Removed 1mL of culture before adding IPTG and at 1-hour intervals after addition of IPTG, until after 5 hours after gene expression induction.
  • Spun down cells for 15 minutes at 5,300 ref at 4 degrees Celsius, and decanted supernatant.
  • Weighed wet pellet using analytical balance.

Julian's Work

• grew a 10mL starter culture by picking a colony from the plate. Grew in LB with Chlor at 1000X dilution. Grew in shaking incubator at 37C
  • grew well in ~12 hours
• Danny and I also met with Mark Arbing:
• Lysis
• Using the Dnase and Lysosyme is ok. We need protease inhibitors too*
• For our lysis buffer it should be high salt (100-150mM), pH balanced (around 7-8), can add some glycerol or other stuff too

we can add some EDTA too but then we need to buffer exchange it before we load it onto the Ni column

• We can use the french press or sonication (just ask him for help or training). They also have like a fancy french press we can use which he recommended if we use a machine for lysis

you still need the lysis buffer for this tough

• Purification
• we can use our gravity column with an IMAC resin and that's fine
• we need to store our protein in a buffer that's not super high salt

so we can elute, dialyze or buffer exchange

• the lysis buffer can be the same as the initial buffer for the column though the lysis shouldn't have any imidizole
• if that column doesn’t work well we can ask to use his HPLC machine
  • they will run it for us
  • we need to provide the sample (1-10mL for SEC)
  • we need to provide a buffer too
  • I think SEC is better than trying to do ion exchange