Team:Heidelberg/Notebook/Killing II/Cloning


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Killing II - Cloning Strategy

For the prey-killer system two different parts were necessary. A sender part (BBa_K150000) containing LuxI and three killer parts containing LuxR and different colicin operons controlled by PLuxR promoter. The sender part was designed from BBa_J23107 and BBa_F1610 and the three killing parts were designed from BBa_T9002, the pColE1 and the pColE9-J plasmid. The figures below show a detailed cloning strategy.

Figure 1 Cloning strategy of the sender part (BBa_K15000) Parts BBa_J23107 and BBa_F1610 were combined according to the BioBrickTM assembly. Therefore BBa_J23107 on BBa_J61002 was digested with SpeI and PstI and BBa_F1610 was digested with XbaI and PstI. Cloning was finished with the final ligation.
Figure 2 Cloning strategy of the LuxR-colicin-receiver parts (E1: BBa_K150009, E9 long: BBa_K150011, E9 short: BBa_K150013) First of all the LuxR receiver part (BBa_T9002) was amplified by PCR. The product, containing a Ptet promoter, the luxR gene followed by the PluxR promoter and a ribosome binding site was cloned into pSB1A2 vector. This vector was extracted from BBa_T9002. In a second cloning step the different colicin operons, amplified from pColE1 or pColE9-J were cloned behind the PLuxR promoter.