Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk/12 August 2015"
(→Multiple Mini Expressions: Optimizing protein yield) |
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#Add 50 ul of starter culture to 5 ml of LB starting around 5 pm along with 5 ul of Kan. | #Add 50 ul of starter culture to 5 ml of LB starting around 5 pm along with 5 ul of Kan. | ||
#Add in the varying amounts of IPTG and use the two different temperatures. | #Add in the varying amounts of IPTG and use the two different temperatures. | ||
+ | #Use the following protocol to analyze the cell pellet. | ||
+ | #Record the weight of the pellets. | ||
+ | #*In total, the pellet weighed 1.75 grams, or about 0.6 grams per 100ml, which is lower than the 0.8 grams we have gotten in the past. | ||
+ | #Transfer pellet to one falcon tube. | ||
+ | #Resuspend in 5 ml/g (8.75ml) of pellet Bug Buster (1x) by pipetting and gently vortexing. | ||
+ | #Put on shaker or rotating mixer for 15 min at RT. | ||
+ | #*Take first fraction, (F1) of this full cell lysate. | ||
+ | #Centrifuge 16000 g 20 min at 4 degrees C | ||
+ | #*Take second fraction of this supernatant (S1) which should contain soluble proteins, while our desired protein should be in the pellet. | ||
+ | #Resuspend in the same volume of 1X Bugbuster 8.75ml as above. | ||
+ | #*Make sure the pellet is completely resuspended! | ||
+ | #*Take Fraction at this point (F2) Cell lysate #2 | ||
+ | #Add DNAse (1 ul) and let rotate 20 min. (This may have been too much DNAse but it should be ok.) | ||
+ | #Add dry lysozyme to concentration of 200 ug / ml | ||
+ | #*Dissolved the lysozyme in water and added the water. | ||
+ | #*Let incubate on ice 30 minutes, swirl every 5 min. | ||
+ | #Take another fraction (F3) here, to see if there is a difference after adding the DNAse and the lysozyme. | ||
+ | #Add 6 volumes of 1:10 diluted bugbuster (.1X) | ||
+ | #*Can split up into two falcon tubes if necessary. | ||
+ | #*Vortex for 1 minute | ||
+ | #Centrifuge 16000g 15 min. 4 degrees C to collect inclusion bodies. | ||
+ | #Collect supernatant as fraction (S2). Inclusion body should be the pellet. | ||
+ | #Resuspend inclusion body pellet in 3% SDS solution and incubate at 60 C in the water bath for 2 hours. | ||
+ | #*Used 10 ml of SDS. (Final) | ||
+ | #Store at 4C for further processing and analysis. |
Revision as of 22:20, 20 August 2015
Multiple Mini Expressions: Optimizing protein yield
- Today we are testing multiple growth conditions of our bl21 cells for total cell and protein yield. Parameters we are optimizing are iptg concentration, and growth temperature.
IPTG concentration/Temp | 37C | 30C |
---|---|---|
0.1 mM | ||
0.5 mM | ||
1.0 mM | ||
2.0 mM |
- We will test these different conditions using the following protocol
- Grow up a starter culture using glycerol stock #1 of our silk in BL21 to an OD of around 1. (4 to 5 hours)
- Add 50 ul of starter culture to 5 ml of LB starting around 5 pm along with 5 ul of Kan.
- Add in the varying amounts of IPTG and use the two different temperatures.
- Use the following protocol to analyze the cell pellet.
- Record the weight of the pellets.
- In total, the pellet weighed 1.75 grams, or about 0.6 grams per 100ml, which is lower than the 0.8 grams we have gotten in the past.
- Transfer pellet to one falcon tube.
- Resuspend in 5 ml/g (8.75ml) of pellet Bug Buster (1x) by pipetting and gently vortexing.
- Put on shaker or rotating mixer for 15 min at RT.
- Take first fraction, (F1) of this full cell lysate.
- Centrifuge 16000 g 20 min at 4 degrees C
- Take second fraction of this supernatant (S1) which should contain soluble proteins, while our desired protein should be in the pellet.
- Resuspend in the same volume of 1X Bugbuster 8.75ml as above.
- Make sure the pellet is completely resuspended!
- Take Fraction at this point (F2) Cell lysate #2
- Add DNAse (1 ul) and let rotate 20 min. (This may have been too much DNAse but it should be ok.)
- Add dry lysozyme to concentration of 200 ug / ml
- Dissolved the lysozyme in water and added the water.
- Let incubate on ice 30 minutes, swirl every 5 min.
- Take another fraction (F3) here, to see if there is a difference after adding the DNAse and the lysozyme.
- Add 6 volumes of 1:10 diluted bugbuster (.1X)
- Can split up into two falcon tubes if necessary.
- Vortex for 1 minute
- Centrifuge 16000g 15 min. 4 degrees C to collect inclusion bodies.
- Collect supernatant as fraction (S2). Inclusion body should be the pellet.
- Resuspend inclusion body pellet in 3% SDS solution and incubate at 60 C in the water bath for 2 hours.
- Used 10 ml of SDS. (Final)
- Store at 4C for further processing and analysis.