Team:Heidelberg/Notebook/Killing II/Cloning

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Revision as of 02:15, 29 October 2008

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 P_LuxR 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.

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 ==Killing II - Cloning Strategy==
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+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 P<sub>LuxR</sub> 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.
+[[Image: Fig_3-cloningstrategy_sender.png | center |thumb|900px|''' 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.  ]]
+[[Image: Fig_4-cloningstrategy_colicinreceivers_small.png | center |thumb|900px|''' 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.  ]]