IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/03

UNAM-Genomics-Mexico team

Main project page Previous entry      Next entry

Working on LovTAP fused to promoters:Ligations to backbone pSB3K3 ans pSB1C3 Once the plasmid pSB3K3 was correctly digested with the enzymes EcoRI and PstI, I have started the dephosphatation reaction in order to prepare it for ligation with promoters-LovTAP and BBa_K098991(cI regulated promoter+RBS+GFP) constructions. Dephosphatation mixture Reactive Quantity DNA 15μL Buffer 10X 3μL Dephosphatase Enzyme 1.5μL HPLC Up to a final volume of 30 μL of the mixture (10.5μL) Procedure 1.Incubate the samples at 37°C during 20 min. 2.Incubate the samples at 65°C during 10 min. Ligation Procedure:LovTAP-Promoters and BBa_K098991 to plasmid pSB3K3 and pSB1C3 1. Prepare the ligation mixture taking into account the quantity of the DNA inserts -LovTAP+Promoters and BBa_K098991- and the receiver DNA -plasmid pSB3K3/pSB1C3-. This can be check in the following gel. Ligations LovTAP+promoters and BBa_K098991 to plasmid pSB3K3..Lane1:Ladder.Lane2:Plasmid pSB3K3 (EcoRI/PstI)dephosphatated replica 1.Lane3:Plasmid pSB3K3 (EcoRI/PstI)dephosphatated replica 2.Lane4: Plasmid pSB1C3 (EcoRI/PstI)dephosphatated .Lane5:Plasmid LovTAP+J23102 (EcoRI/PstI).Lane8: Plasmid LovTAP+J23105 (EcoRI/PstI).Lane9:Plasmid LovTAP+J23114 (EcoRI/PstI).Lane10:Plasmid LovTAP+J23117 (EcoRI/PstI).Lane11:LovTAP Mr.Gene PCR amplified product (EcoRI/PstI). Lane12:BBa_K098991 (EcoRI/PstI. Ligation mixture using plasmid pSB3K3 Reactive Quantity DNA insert (LovTAP-J23117, LovTAP-J23105, LovTAP-J23114, LovTAP-J23102 and K098991) 6μL DNA plasmid (pSB3K3) 3μL Buffer 10X T4 Ligase 2μL Enzyme T4 Ligase 1μL HPLC Up to a final volume of 20 μL of the mixture (8μL) Ligation mixture using plasmid pSB1C3 Reactive Quantity DNA insert (LovTAP PCR amplified product from Mr.Gene) 6μL DNA plasmid (pSB1C3) 2μL Buffer 10X T4 Ligase 2μL Enzyme T4 Ligase 1μL HPLC Up to a final volume of 20 μL of the mixture (8μL) 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 LovTAP ligated to each promoter and BBa_K098991, if the ligation was correctly done. Working on cI inverter construction and fusion to pSB3K3 backbone. LovTAP repressor activity reporter system Once the plasmids harboring the parts showed in the next table were correctly isolated, I am going to digest P0451(RBS+cI repressor) with EcoRI and SpeI and K098991 (cI regulated promoter+RBS+GFP) 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. The plasmid harboring J23101 promoter will be digested with SpeI and PstI. Part Lenght K098991 (cI regulated promoter+RBS+GFP) 933 bp P0451(RBS+cI repressor) 930bp promoter J23101 38 bp Restriction enzymes EcoRI and SpeI Plasmid digested: Plasmid harboring P0451 isolated from colony 6. Restriction enzymes XbaI and PstI Plasmid digested: Plasmid harboring K098991 isolated from colony 8. Restriction enzymes SpeI and PstI Plasmid digested: Plasmid harboring promoter J23101. 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 20 min. Results restriction enzyme assays Quantity loaded 3μL Restriction enzyme assays. Lane1:Ladder. Lane2:Plasmid pS1C3. Lane3:Plasmid harboring promoter J23101 (SpeI/PstI).Lane4:P0451 colony 6 (EcoRI/SpeI) .Lane5: K098991 colony 8 (XbaI/PstI). The sizes of the digested products are around the expected lenght for each part. Working on Lumazine synthesized from Mr.Gene I am going to do PCR reactions in order to amplify the Lumazine construction synthesized. The product obtained will be purified using the High Pure PCR Product Purification kit from Roche. Primers: Forward:Preffix primer Reverse:Suffix primer PCR template: Plasmid harboring Lumazine from Mr.Gene Reaction 1 Reactive Quantity MgCl2 3μL Buffer 3.3X 6μL dNTP's 3μL (0.4mM) Primer Forward 2.5μL (5pmol/μL) Primer Reverse 2.5μL (5pmol/μL) DNA template(Mr.Gene Plasmid Lumazine 1μL + 9μL HPLC) 1μL HPLC Up to a final volume of 30 μL of the mixture Reaction 2 Reactive Quantity Buffer 3.3X 9μL RTTH enzyme 0.5μL HPLC Up to a final volume of 20 μL of the mixture 35 PCR programmed cycles: The reaction starts at 95°C during 5 min. After this step, the reaction 2 is loaded to the reaction 1 sample. Each cycle is programmed as follows: Temperature Time 94°C 45s 60°C 45s 72°C 1 min After the cycle 35, the temperature changes to 72°C during 10 min and ends at 4°C. Results: Lumazine PCR from Mr. Gene Lumazine construction was well amplified. The PCR product from each replica was around the expected size 805nt. Lumazine Mr.Gene PCRs. Lane1:Ladder. Lane4: Lumazine PCR. Lane5:Lumazine PCR. The other lanes are samples from other experiments