Team:Hawaii/Shelby

Shelby's Research Journal

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DH5-α Transformation

June 5th, 2018

An RFP BioBrick from the iGEM kit plate #3 (part BBa_E1010) was used in the DH5-α transformation of competent E. coli cells for protein expression. The RFP BioBrick was resuspended using 10µl of milliq H20 and left to sit under the hood for 10 minutes. Protocol was followed using the DH5-α Transformation protocol (described in the iGEM lab notebook). 1µl of resuspended RFP was used. Colonies were heat shocked in a 42 ºC water bath for 45 seconds, and then placed on ice for 2 minutes. Since the RFP BioBrick is in plasmid PSB1C3, chloramphenicol was used as the antibiotic in our LB-agar petri dish to select for colonies that express the insert. 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 at ~4:30 pm on 6/4/2018. Upon checking plates at 1pm on 6/5/2018, 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.