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

• Overnight culture of transformants
• Restriction enzymes EcoR1 and Pst1

Protocol

• Plasmid extraction of transformants
• Single (EcoR1) and double (EcoR1 and Pst1) restriction digest of plasmid extracts
• Run digests on a gel

Results

Figure 1. Gel electrophoresis of the single and double digests using EcoR1 and Pst1.

• Lane 1-7: Single digests of colonies 2-8 in order
• Lane 8: 1 Kbp Molecular weight marker
• Lane 9-15: Double digests of colonies 2-8 in order

Discussion

This gel shows that colony 2 contains only pSB1AT3 (3.5 Kbp) and that the rest contain pSB1AT3 (3.5 Kbp) with an RFP (1 Kbp)insert (total 4.5 Kbp). Also please note that the gel is in fact off angle due to being twisted and the picture not being straight on. This is as was shown in the other tests that were performed this week.

Conclusion

We can conclude from these results that our ligation attempt has failed and that in order to carry on the construction we would need to re-perform the ligation using more of the PCR product. However this will not be done for the moment for these reasons: Firstly we believe that we can use another BioBrick already in the registry that will suit our needs, this is only made possible by the looming prospect of using Gibson cloning method to construct our Bricks; Secondly there are other Bricks that our more important for our project that we need to concentrate on.