Team:UCLA/Notebook/Protein Cages/29 July 2015

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Phillip's notes:

Introduction: It has been decided that a new gBlock will be ordered. After some consultation, it has been decided that the gBlock will be designed with an additional handle for primer annealing. Regarding the protein expression, the lysis be done and protein concentration will be measured today.

Procedures:

Designing the construct: The gBlock to be ordered is titled “PCquad Optimized for iGEM 3.0.” Using a random sequence generator, http://faculty.ucr.edu/~mmaduro/random.htm the handles were designed to be 30 bp. An initial primer was designed and checked using IDT Oligoanalyzer for secondary structure, and non-specific binding within the construct. The sequence was optimized by changing some bases. The highest deltaG was -9kcal/mol for non-specific binding between the forward primer and the reverse complement of the cage. The Reverse primer was designed in a similar method. The highest deltaG was -8kcal/mol for non-specific binding between the reverse primer and the forward sequence of the cage.

Picking the insertion mutant: Mutant number 10 was chosen due to the site’s potential accessibility by thrombin protease.

Cell lysis:

The 0.093g cell pellet was lysed in 1mL of lysis buffer. Lysis buffer composition: 50mM phosphate pH 8 300mM NaCl 10mM imidazole 1 protease inhibitor tablet/10mL 100ug/mL 2uL DNase/mL

The lysis buffer was added to the cell pellet, and a pipette was used to resuspend the pellet. The tube was vortexed for 20 seconds in 5 second intervals, and then iced for 5 minutes. This was repeated three times. The solution was then spun down for 10min at max speed, and the supernatant was removed.

Observations: Upon adding lysis buffer, the solution appeared very milky/cloudy. Perhaps too much cells were used. The final supernatant had a moderate yellow tint. Both the pellet, and the supernatant were stored in the -20C. It is possible that the pipettes used may be erroneous.

Conclusions: BCA will be used to measure protein concentration tomorrow, in order to determine how much of the His-resin to use.