Experimental Flow

Below we have outlined the path our project took, from the initial cloning through experimentation and submission.

Isolated LacZ from iGem part BBa_K564012

1) Rehydrated DNA from distribution kit

2) Transformed into DH5a chemically competent cells

3) Extracted plasmid

4) PCR mutagenesis to engineer desired cut sites around LacZ

Transferring LacZ into pET28

1) Digested pET28 and new version of BBa_K564012 (with added cut sites)

2) Ligated together

3) Submitted for sequencing to confirm

4) Transformed into new DH5a chemically competent cells

Creation of delta-M15 mutant

1) Deletion of segment including alpha fragment of LacZ using NEB Q5 site directed mutagenesis kit

2) Submitted for sequencing to confirm successful deletion

Proximity dependent ligation assay

1) Probe segments and bridge segments mixed with T4 ligase in presence or absence of thrombin

2) Gel electropheresis to detect ligation

Cloned delta-M15 mutant into submission vector

1) Digested linearized pSB1C3 with our mutant

2) Ligated together

3) Transformed into new DH5a chemically competent cells

5) Extracted DNA

6) Dried, packaged, shipped

Protocols

Below are the protocols we used for al stages of our project. We wrote them to be as easy to follow as possible, to accommodate new team members with little lab experience.W= We hope future teams find these highly detailed versions of common protocols useful.