Team:TUDelft/SDP Results
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=='''I-SceI homing endonuclease'''== | =='''I-SceI homing endonuclease'''== |
Revision as of 22:05, 21 October 2009
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'
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 works as expected.
As shown in the cloning strategy, the restriction site was cloned inbetween the promoter and the RBS, resulting in , which was expected not to influence transcription. However, when a culture containing this biobrick was induced with anhydrotetracyclin (aTc), no fluorescence was observed. This led to a modification of the cloning strategy.
A simpler part was constructed (), which only contained the restriction site in front of the aTc inducible GFP-LVA generator.
This part was successfully sequenced and shown to work. Firstly, aTc fluorescence induction worked, as shown in the following image.
Secondly, the part was digested with PstI alone and with PstI and I-SceI. The lenght of the entire biobrick was expected to be 5095bp. The second lane in the adjacent image shows that the observed length corresponds to the predicted length. The part was also digested with PstI and I-SceI, theoretically resulting in two bands of 3204 and 1891bp. The resulting bands in lane 3 of the same figure show that these also correspond well.
Thirdly, the sequence was confirmed by sequencing the part in forward and reverse direction.
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