Cloning Strategy

Conjugation Testing Plasmid1 K175005

Conjugation Testing Plasmid2 K175006

R751 Plasmid - Knockouts

TrbK knockout

TrbK knockout

OriT-R knockout

OriT-R knockout

TrbC knockout

TrbC knockout

Constructing the Self Destructive plasmid

Next figures will illustrate how the self destructive plasmid will be constructed.

Figure 6: Assembly of GFP-LVA and ribosomal binding site.
A weak ribosomal binding site and a GFP which contains a LVA tag for rapid degradation are cloned into a high copy BioBrick assembly plasmid.

Figure 7: Assembly of GPF-LVA-ribosomal binding site with double terminator.
A double terminator is added to the GFP-LVA -ribosomal binding site construct and cloned into a high copy BioBrick assembly plasmid.

Figure 8: Assembly of tetR promoter and I-SceI restriction site.
Since the restriction site of I-SceI is not available as BioBrick, this restriction site should be standardized as BioBrick. Therefore we synthesis this restriction site flanked with the BioBrick prefix an suffix. This restriction site is than cloned into a high copy BioBrick assembly plasmid. After standardizing the restriction site this restriction site is combined with a TetR repressible promoter and cloned into a high copy BioBrick assembly plasmid. This tetR promoter will control transcription of the I-SceI endonuclease, in order to avoid constantly transcription of the I-SceI.

Figure 9: Assembly of I-SceI and TetR
The tetR promoter which controls the GFP-LVA transcription is repressed by TetR. Therefore the SDP should contain a tetR gene as well in order to control GFP-LVA transcription. The I-SceI restriction site and TetR generator are cloned into a high copy BioBrick assembly plasmid.

Figure 10: Assembly of endonuclease, tetR promoter and I-SceI restriction site
At this point all the basic parts of the construct are built. In order to construct the SDP these parts should be reassembled. The I-SceI generator(I-Sce-I gene under control of a Lac promoter) is assembled with tetR promoter and I-SceI restriction site and cloned into a high copy BioBrick assembly plasmid.

Figure 11: Assembly of GFP-LVA construct and TetR construct.
The constructs showed in figures 7 and figure 9 are assembled and cloned into a high copy BioBrick assembly plasmid.

Figure 12: Final assembly of the SDP.
At this point all the constructed biobricks are assembled together in order to construct the final construct, the Self Destructive Plasmid.