LacI BioBrick Construction

Aims

• To use PCR to extract lacI (promoter, ribosome-binding site (RBS) & coding sequence (CDS)) from plasmid pMutin4 and ligate into vector pSB1AT3 in front of red fluorescent protein (RFP).

Materials

• Seven overnight cultures
• Restriction enzyme EcoR1

Protocol

• E. coli DH5α was transformed again with our ligation mix.
• The seven overnight cultures underwent plasmid extraction.
• The seven plasmid extracts were then restriction digested with EcoR1 to linearise.
• The seven digests were then run on a gel.

Results

This procedure was followed in order to determine whether our lacI had been ligated into the plasmid vector.

Figure 1: Gel electrophoresis of the seven plasmid extracts which have been digested with EcoR1.

The gel shows bands in lanes:

• 2-3 (samples 1-2) that correspond to vector only (3.5 Kbp) and
• 4-8 (samples 3-7) that correspond to the size of pSB1AT3 plus the original RFP insert (4.5 Kbp)

Conclusion

The conclusion that can be drawn from this gel is that the lacI has not been inserted into the plasmid as we desired. However due to some confusion as to the actual size of the vector a set of double digests will be performed tomorrow to try to extract insert form the plasmid if any is present.