Team:TUDelft/SDP Overview


Module 2: Self Destructive Plasmid

The message that the helper plasmid caries and is delivered by the donor cell to the acceptor cell should disappear in the donor cell, after being passed to the acceptor cell. Therefore we would like to build a plasmid which is able to destruct itself. Therefore the plasmid should be able to degrade itself. This degradation should be inducible. In order to facilitate the degradation of the plasmid we construct a plasmid whit a I-SceI endonuclease gene which is under control of a Lac promoter. This plasmid also contains several restriction sites of I-SceI in order to start degradation after inducing I-SceI transcription. In order to construct the Self Destructive Plasmid (SDP) we build the construct showed in Figure 5 and clone it this construct into a high copy BioBrick assembly plasmid.
Construct SD.jpg
Figure 5: The construct to be build and cloned into a high copy BioBrick assembly plasmid.

P-lacI.jpgp-lacII-SceI restriction site.jpgI-SceI restriction site
P-tetR.jpgp-tetRGFP with LVA tag.jpgGFP with LVA tag
Constitutive promoter.jpgConstitutive promoterTetR.jpgTetR
Ribosomal binding site.jpgRibosomal binding siteI-SceI.jpgI-SceI

In order to build this construct 6 other constructs should be built, assembly of these constructed will result to the above showed construct. We like to use in the first place already existing BioBricks. Since there is no I-SceI restriction site available as BioBrick, we should standardize this restriction site. The half-life time of the GFP-LVA is 40-80 minutes. This short half-life is useful for this project. After constructing the SDP we would like to induce the transcription of this GFP and stop the induction. At the time that the signal of the GFP disappears we induce the transcription endonuclease and in order to check if the plasmid is degraded we induce the transcription of the GFP once again.

For more information on the cloning strategy of constructing self destructive plasmid check the cloning strategy page

Checking the Self Destructive Plasmid

In order to check the activity of SDP some experiments should be done. During the assembly of the construct several control experiments will be done. After building a construct, the construct will be checked by PCR and agarose gel electrophoresis to check the length of the construct. Verification forward primer(VF2) and verification reverse (VR) will be used. The expression and activity of I-SceI will be checked by an Assay. The construct showed in figure.10 will be used to check the expression of I-SceI. The same plasmid or other vectors containing I-SceI recognition site could be used to check the activity of the endonuclease. Incubating this plasmid with expressed and purified I-SceI and agarose gel electrophoresis will show if the endonuclease is active. These results will be compared with activity of commercial available I-SceI homing endonuclease. The expression of TetR will be checked by expression TetR from the in figure.9 showed construct. Western Blot analysis should show if TetR is expressed.