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

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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.



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