IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/02

UNAM-Genomics-Mexico team

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Working on cI inverter construction, pSB3K3 backbone and E0240 part Once the plasmids harboring the parts showed in the next table were correctly isolated, I am going to digest some with EcoRI and PstI and the others with XbaI and PstI in order to fuse them with their corresponding part. Part Lenght K098991 (cI regulated promoter+RBS+GFP) 933 bp P0451(RBS+cI repressor) 930bp E0240 (RBS+GFP protein) 876 bp J04450 pSB3K3 backbone 1010 bp Restriction enzymes EcoRI and PstI: K098991 preparation for ligation with plasmid pSB3K3 and J04450 preparation for ligation with promoters-LovTAP, trpL-cI inverter and trpL-GFP constructions for characterization. Plasmids used: Plasmid harboring K098991 isolated from colony 8. Plasmid harboring J04450 isolated from colony 8. Two replicas Reactive Quantity Plasmid DNA 15μL Buffer 2 4μL BSA 1μL Enzymes 2μL for each one. HPLC Up to a final volume of 40 μL of the mixture (16μL) Restriction enzymes XbaI and PstI: P0451 and E0240 preparation for ligation with constitutive promoter (J23101) and trpL promoter Plasmids used: Plasmid harboring P0451 isolated from colony 6. Plasmid harboring E0240 isolated from colony 5. Two replicas Reactive Quantity Plasmid DNA 15μL Buffer 2 4μL BSA 1μL Enzymes 2μL for each one. HPLC Up to a final volume of 40 μL of the mixture The reactions were incubated at 37°C overnight. Note: Inactivate the enzymes at 80°C during 10 min. Results restriction enzyme assays Restriction enzyme assays: EcoRI/PstI and XbaI/PstI. Lane1:Ladder. Lane2:J4450 colony 8 replica 1 (EcoRI/PstI). Lane3:J4450 colony 4 replica 2 (EcoRI/PstI).Lane4: E0240 colony 5 replica 1 (XbaI/PstI). Lane5: E0240 colony 5 replica 2 (XbaI/PstI).Lane6:P0451 colony 6 (XbaI/PstI) .Lane7: K098991 colony 8 (EcoRI/PstI). The sizes of the digested products are around the expected lenght for each part.