Team:Hawaii/Shelby

Shelby's Research Journal

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DH5-α Transformation

June 5th, 2018

An RFP BioBrick from the iGEM kit plates (part BBa_E1010) was used in the DH5-α transformation of competent E. coli cells for protein expression. This BioBrick has been used 360 times as of 6/4/2018, per the iGEM page describing this part. This RFP BioBrick is 25.4237 kDa. Its excitation peak is 584 nm, and its emission peak is 604 nm. This BioBrick is also optimized for bacterial expression, with a Codon Adaptation Index (CAI) of 0.84 for E. coli. Transformed cells were transferred to four LB-Chloramphenicol plates and left to incubate at 36 ºC for about 20 hours. After incubation, no visible colonies were observed. It appears that our colonies did not take up the plasmid. Our next steps will be to see whether iGEM has their own transformation protocol when inserting BioBricks.

June 8th, 2018

A second transformation was attempted in an effort to obtain transformed colonies containing the RFP BioBrick BBa_E1010. After incubating for ~30 hours, the second transformation yielded cell growth on both the positive control plate and our RFP BioBrick plate. In this second transformation, four plates were prepared: One low-concentration RFP transformation, one high-concentration RFP transformation, one low-concentration (+) control, and one high-concentration (+) control. The DNA used for the positive control was BioBrick BBa_J04450, which also codes for an RFP protein. Only the high concentration plates yielded cell growth, while the low-concentration plates yielded no growth. Our (+) control plate yielded about 3-4 colonies, with one large group of cells growing together in the middle of the plate. All colonies in the (+) control plate were red in color and glowed red under UV light. Our RFP transformation yielded cell growth (2 colonies), but were not red in color and did not glow under UV light. This indicates that our cells took in the plasmid DNA, since they were growing successfully on the chloramphenicol plate, but did not produce RFP proteins. The part used for RFP transformation (part Bba_E1010) was recorded as being E. coli optimized. It is possible that this BioBrick does not contain a promoter. The next steps will be to confirm presence of both BioBricks.