Difference between revisions of "Team:UCLA/Notebook/Protein Cages/14 July 2015"

 
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Phillip's notes:
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Phillip's notes
  
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Introduction:  Inoculation into the large culture, and protein induction, will be done today.
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Introduction:  Cells will be harvested, and cell lysis will be done if reagents are available.
  
 
Procedures:
 
Procedures:
  
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Inoculation:
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Harvesting of cells:
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2mL of the starter culture was given to Tyler in order to miniprep.  The starter culture was taken out at 12:00PM at left on the bench while autoclaving a 2L flask.  Upon cooling the flask, 1L of LB and 1mL of 100mg/mL carbenicillin was added to the flask.  The remaining 8mL of starter culture was poured into the flask at 1:30PM.   
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The 2L flask was taken out of the 30C incubator at 12:00PM, and left on the bench for about 2.5 hours.  The cells appeared to be dense, as indicated by the milky-yellow color of the culture.  Four 250mL centrifuge bottles were autoclaved, along with 1L of ddH2O.  The rotor used for centrifugation was cooled in the cold room before use.  Upon cooling the bottles and the sterile water, the liquid culture from the 2L flask was distributed evenly between three bottles (and balanced by adding sterile water).  The bottles were centrifuged at 6000g for 15 minutes, and the supernatant was discarded.  Each cell pellet was resuspended in 25mL of sterile water, and vortexed to resuspend.  Each bottle was pooled, mixed, and distributed evenly between two 50mL falcon tubes.  8mL from one was taken to be used for a small-scale protein purification.  The remaining bottles were balanced, and centrifuged again for 15min at 6000g.  The supernatant was discarded, and the pellet was stored in the -20C freezer.  The sample for small-scale purification was centrifuged, resuspended, and transferred to a 1.5mL before collecting the pellet to be frozen at -20C.
  
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Induction:
 
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At 3:30PM (2 hours) 0.0477g of IPTG was dissolved in 1mL of LB, and added to the flask for a total concentration of 0.2mM.  The OD600 was measured to be 0.165.  Because there was no access to an 18C incubator, the flask was left in the 30C incubator.
 
  
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Observations:  OD600 should have been measured before induction, as the ideal OD600 at time of induction should be 0.6.  Also, the flask should have been sterilized the day before in order to not have to leave the bacteria culture on the bench.
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Conclusions:  Since lysis cannot be continued until DNase, protease inhibitors, and lysozyme is obtained, it will be put on halt for the time being.

Latest revision as of 19:08, 17 July 2015

iGEM UCLA




Intro: Pondered how to express cage in iGEM compatible vector. Designed and ordered appropriate primers.

Vinson had a plasmid that contains: T7 promoter-RBS-iGEM suffix. After much thought, I designed primers to amplify our g block with the iGEM prefix, and tack on a his tag, stop codon, and the suffix in two rounds of PCR. The forward primer will be the iGEM prefix and the first 20 nucleotides of the cage sequence. The first reverse primer will be the last 20 nucleotides of the cage and the his tag. The second reverse primer will inclund the final nucleotide of the cage, his tag, stop codin and suffix. This two step addition was the only way to add the his tag and stop codon without going over the 60bp limit imposed by IDT for ordering oligos. Sri said to express the cage in this new iGEM compatible vector, as well as the pET-22b vector in parallel, just to see which is better. He recommended that we initially transform the ligated plasmids into DHSalpha competent cells (in his lab) to verify correct plasmids before finally transforming them into BL21-DE3 competent cells for protein expression.

Tyler Lee --Wtleeiv 23:00, 14 July 2015 (CDT)



Phillip's notes

Introduction: Cells will be harvested, and cell lysis will be done if reagents are available.

Procedures:

Harvesting of cells: The 2L flask was taken out of the 30C incubator at 12:00PM, and left on the bench for about 2.5 hours. The cells appeared to be dense, as indicated by the milky-yellow color of the culture. Four 250mL centrifuge bottles were autoclaved, along with 1L of ddH2O. The rotor used for centrifugation was cooled in the cold room before use. Upon cooling the bottles and the sterile water, the liquid culture from the 2L flask was distributed evenly between three bottles (and balanced by adding sterile water). The bottles were centrifuged at 6000g for 15 minutes, and the supernatant was discarded. Each cell pellet was resuspended in 25mL of sterile water, and vortexed to resuspend. Each bottle was pooled, mixed, and distributed evenly between two 50mL falcon tubes. 8mL from one was taken to be used for a small-scale protein purification. The remaining bottles were balanced, and centrifuged again for 15min at 6000g. The supernatant was discarded, and the pellet was stored in the -20C freezer. The sample for small-scale purification was centrifuged, resuspended, and transferred to a 1.5mL before collecting the pellet to be frozen at -20C.


Conclusions: Since lysis cannot be continued until DNase, protease inhibitors, and lysozyme is obtained, it will be put on halt for the time being.