Team:UCLA/Notebook/Recombinant Expression/5 August 2015

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12-mer Expression, cont.

The two start up cultures are taken from the shaking incubator and the OD checks are 0.3 and 0.2, respectively. The start-up culture with the higher OD is poured into a L of LB with 1mL of Chloramphenicol. The contents are put into a shaking incubator at 37 degrees Celsius and left to incubate for 3hours. A 1mL sample is taken from the flask, and IPTG is added to a final concentration of 0.5mM. After IPTG addition, a 1mL sample of culture is taken every hour after IPTG addition for 4 hours total. The samples will be used in an SDS PAGE to check for verification of over expression of the 12-mer proteins. The contents are then left to incubate overnight for approximately 17 hours.

Observations

Before IPTG addition, a 1mL sample of culture is extracted form the flask and pipetted into a 1.5mL micro centrifuge tube. The contents are spun with a balance at 3000 rpm for a minute. However, no visible cell pellet appeared at the bottom of the tube. Only LB supernatant was observed. This happened again when a 1mL sample of culture was taken 1 hour after IPTG addition. 2 hours after IPTG addition, a 1mL sample was spun down and a small pellet appeared at the bottom. Two cell pellets were successfully recovered 3 and 4 hours after IPTG addition. When taking the OD after IPTG had been induced into the cells for 4 hours, the OD value was 0.2, significantly lower than expected when compared to previous 9-mer expression. This might have been because IPTG was added 3 hours after initial incubation whereas IPTG was added 4 hours after initial incubation for 9-mer cells. This hour would be significant since E-coli population doubles every 20 minutes. A cell pellet was not seen possibly because the cells were still pretty dilute in the LB growth medium, and the 1mL sample was extracted near the surface of the liquid. (Although a shaking incubator vigorously shakes the entire contents of the flask, a higher concentration of cells would most likely be found near the bottom of the flask.) Also, it's possible that a 12-mer protein, which by definition is three monomer units longer than a 9-mer, requires more time for the cells to assemble and replicate. We will consult the Silk Genetics team on this, and it will be interesting to see if these observations reappear when preparing the 15-mer proteins next week.