Characterization of BBa_I20270

After we participated in the interlab and did not successfully complete the lab work because of lack of one lab measurement equipment, we decided to use parts from the registry kit that was submitted to us from the headquarters to do its characterization. We characterized BBa_I20270 and below is how we did it and the results we got. However, we started the characterization process late and by the wiki freeze we will not have documented all the results as we are still in analysis.

Part Protocols

Testing the IGEM part BBa-120270, a positive control for the interlab measurement study.

Procedure

1. Suspended the plasmid DNA with 10uL of distilled water, left it to stand for 5 minutes.
2. Transformed the competent E.coli BL21 cells with 1uL of the resuspended plasmid DNA.
3. Cultured the transformed cells on LB agar overnight at 37⁰C
4. Transferred one single colony of the transformants to freshly prepared LB broth, 10mL of LB broth
5. Incubated for 6 hours at 37⁰C at 250rpm on a rotary incubator.
6. At an OD>0.5, picked a 1mL aliquot from the cell suspension pre-induction, lysed the cells with chloroform, a drop of chloroform to a milliliter of cell suspension then vortexed to breakdown the cells.
7. Pipetted 100uL of supernatant into a well on a 96 well plate, then determined the absorbance of the green fluorescent protein at 500nm wavelength.
8. Induced expression for 2hours using IPTG, then picked a 1mL cell aliquot, lysed the cells with chloroform and analyzed the supernatant to determine the absorbance at 500nm wavelength.

Challenges

Limited information about the plasmid and gene circuit.

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