Difference between revisions of "Team:UCLA/Notebook/Recombinant Expression/19 May 2015"

 
(2 intermediate revisions by 2 users not shown)
Line 277: Line 277:
  
 
* Starter culture failure
 
* Starter culture failure
−
** [https://2015.igem.org/Team:UCLA/Notebook/Recombinant_Expression/18_May_2015 Yesterday's] attempt at creating a starter culture from the 5th plating of the old transformatants did you grow.  We hit an OD600 of 0.004, basically negligible growth.   
+
** [https://2015.igem.org/Team:UCLA/Notebook/Recombinant_Expression/18_May_2015 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.   
 
*** 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.   
 
**** Incubated plates at 37 degrees shaking at 1415.  Will check OD of starter cultures at 1900.   
Line 290: Line 290:
 
** Spun down cells for 15 minutes at 5,300 ref at 4 degrees Celsius, and decanted supernatant.   
 
** Spun down cells for 15 minutes at 5,300 ref at 4 degrees Celsius, and decanted supernatant.   
 
** Weighed wet pellet using analytical balance.
 
** 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:
 +
*<b>Lysis</b>
 +
*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
 +
*<b>Purification</b>
 +
*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

Latest revision as of 23:07, 22 May 2015

  • 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