Team:UCLA/Notebook/Protein Cages/14 July 2015

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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: Inoculation into the large culture, and protein induction, will be done today.

Procedures:

Inoculation: 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.

Induction: 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.

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.