Experimental results

The experimental results for the Self Destructive Plasmid are detailed below. In the process of constructing it, we deviated from the cloning strategy after proving that it didn't work as expected.

I-SceI restriction site

Construction of the I-SceI homing endonuclease restriction site was done by primer synthesis. Primers were annealed and ligated into a pSB1AK3 backbone (3189bp without the insert). Together with the 30bp recognition sequence (shown below) this gave a 3219 bp part. The sites where cleavage of the DNA backbone occurs are indicated with ^.

5' A G T T A C G C T A G G G A T A A^C A G G G T A A T A T A G 3'
3' T C A A T G C G A T C C C^T A T T G T C C C A T T A T A T C 5'

in pSB1AK3 backbone. Lane 1: undigested (supercoiled) plasmid. Lane 2: BglI digested part, expected size = 3219bp. Lane 3: BglI and I-SceI restricted part, expected band lenghts are 2275 and 944bp. Expected lenghts correspond to the bands in the gel.

The resulting biobrick () was successfully sequenced and tested for functionality. Sequencing results can be found on the part page. Functionality was assessed using commercially available I-SceI restriciton enzyme. The plasmid was purified and subjected to a digestion with BglI alone and BglI plus I-SceI. The latter would theoretically result in two fragments of 2275 and 944bp.

The results in the adjecent image show that the digestion indeed results in the expected band pattern. This shows that the restriction site indeed works as expected.

I-SceI homing endonuclease

Integration

Experimental results

Experimental results

'Experimental results'

Experimental results

Experimental results

Experimental results

'Experimental results'

Experimental results

Experimental results

Experimental results

'Experimental results'

Experimental results