IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/25

UNAM-Genomics-Mexico team

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Repeated experiment:Working on J23101 promoter: Ligation to P0451/Lumazine/LuxY Due to the first attempt to ligate P0451, Lumazine and LuxY to promoter J23101 failed, I am going to repeat the experiment. Ligation Procedure:Promoter J23101 with P0451 (RBS+cI repressor)/Lumazine and LuxY 1. 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 J23101-. This can be check in the following gel. Gel for ligations. Lane1:Ladder. Lane2: plasmid harboring the promoter J23101 dephosphatated (SpeI/PstI). Lane3: Lumazine PCR amplified product from Mr.Gene plasmid (XbaI/PstI). Lane4:P0451 colony 6 (XbaI/PstI). Lane5:LuxY purification from Mr.Gene plasmid (XbaI/PstI). Ligation mixture: P0451(RBS+cI repressor) Reactive Quantity DNA insert (P0451:RBS+cI repressor) 4μL DNA plasmid (plasmid harboring the promoter J23101) 2μL Buffer 10X T4 Ligase 2μL Enzyme T4 Ligase 1μL HPLC Up to a final volume of 20 μL of the mixture (11μL) Ligation mixture: Lumazine/LuxY Reactive Quantity DNA insert (Lumazine/LuxY) 5μL DNA plasmid (plasmid harboring the promoter J23101) 2μL Buffer 10X T4 Ligase 2μL Enzyme T4 Ligase 1μL HPLC Up to a final volume of 20 μL of the mixture (10μL) 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. Repeated experiment:Working on cI inverter construction and fusion to pSB1T3 backbone. LovTAP repressor activity:reporter system Due 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 ligate them in plasmid pSB1T3. Ligation Procedure: P0451 + K098991 to plasmid pSB1T3 1. Prepare the ligation mixture taking into account the quantity of the DNA insertS -K098991 and P0451- and the receiver DNA -plasmid pSB1T3-. This can be check in the following gel. Quantity loaded 3μL. Restriction enzyme assays. Lane1:Ladder. Lane4:Plasmid pSB1T3. Lane11:P0451 colony 6 (EcoRI/SpeI) .Lane12: K098991 colony 8 (XbaI/PstI). The other lanes are samples from other experiments Ligation mixture 1 Reactive Quantity DNA inserts (K098991 and P0451) 4μL each DNA plasmid (pSB1T3) 2μL Buffer 10X T4 Ligase 2μL Enzyme T4 Ligase 1μL HPLC Up to a final volume of 20 μL of the mixture (7μL) Ligation mixture 2 Reactive Quantity DNA inserts (K098991+ P0451) 5μL each DNA plasmid (pSB1C3) 3μL Buffer 10X T4 Ligase 2μL Enzyme T4 Ligase 1μL HPLC Up to a final volume of 20 μL of the mixture (4μL) 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.