IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/28
 UNAM-Genomics-Mexico team
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Working on cI inverter construction and fusion to pSB3K3 backbone. LovTAP repressor activity reporter systemOnce the plasmids harboring the parts showed in the next table were correctly isolated, I am going to digest P0451(RBS+cI repressor) with SpeI and PstI, this will be ligated to the Part K098991 (cI regulated promoter+RBS+GFP) digested with XbaI and PstI in order to fuse them to construct the whole cI inverter. Then it will be ligated to trpL and J23101 promoters.
Plasmid digested: Plasmid harboring P0451 isolated from colony 6.
Note: Inactivate the enzymes at 80°C during 20 min. Repeated experiment:Working on cI inverter construction and fusion to pSB1C3 backbone. LovTAP repressor activity:reporter systemDue to the first attempt to ligate K098991 (cI regulated promoter+RBS+GFP) and P0451(RBS+cI repressor) inside plasmid pSB1C3 failed, I am going to repeat experiment. Ligation Procedure: P0451 + K098991 to plasmid pSB1C31. Prepare the ligation mixture taking into account the quantity of the DNA insertS -K098991 and P0451- and the receiver DNA -plasmid pSB1C3-. This can be check in the following gel. Quantity loaded 3μL. .JPG) 
2. Incubate the sample at 16°C overnight. 3. Transform the cells. Click here for the protocol. 4. Culture the cells in the proper selective medium, in our case LB + Kanamycin. 5. Incubate the petri dishes at 37°C overnight. 6. Re-culture the resultant colonies in the proper selective medium; incubate them at 37°C overnight. 7. Analyze the colonies with Colony PCR to confirm that they contain the correct ligation The primers that I am using are: Forward (5'->3'): Preffix primer. Reverse:(5'->3'): Suffix primer. These primers would amplify the whole cI inverter, if the ligation was correctly done. Repeated experiment:Working on J23102 promoter: Ligation to P0451/Lumazine/LuxYDue to the first attempt to ligate P0451, Lumazine and LuxY to promoter J23102 failed, I am going to repeat the experiment. Ligation Procedure:Promoter J23102 with P0451 (RBS+cI repressor)/Lumazine and LuxY1. Prepare the ligation mixture taking into account the quantity of the DNA insert -P0451 (RBS+cI repressor),LuxY and Lumazine- and the receiver DNA -plasmid harboring the promoter J23102-. This can be check in the following gel. 
2. Incubate the sample at 16°C overnight. 3. Transform the cells. Click here for the protocol. 4. Culture the cells in the proper selective medium, in our case LB + Kanamycin. 5. Incubate the petri dishes at 37°C overnight. 6. Re-culture the resultant colonies in the proper selective medium; incubate them at 37°C overnight. 7. Analyze the colonies with Colony PCR to confirm that they contain the correct ligation The primers that I am using are: Forward (5'->3'): Preffix primer. Reverse:(5'->3'): Suffix primer. These primers would amplify LuxY, Lumazine and P0451:RBS+cI repressor plus promoter J23101, if the ligation was correctly done.
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