Team:UIUC-Illinois/Project/Lambda System

Initial transformation of the shuttle construct into our genetically engineered strain containing our helper construct results in integration of the shuttle vector into the attB site. We can reason this out on the above diagram by nothing that the integrase machinery on the right side of the concept is on and results in integration. However, pay close attention to the constitutive promoter in the shuttle construct and keep track of what it does upon integration.

Upon integration, the constitutive promoter turns off the integrase machinery by up regulating the CI repressor upstream of the integrase. This design obtains goal number 3, ensuring integration at a single copy. In this integrated orientation, our inducible promoter (in the modular region), now controls the excision machinery on the left. This integrant will be stable until the excision machinery is induced.

Now we will retrieve the shuttle construct. We add our inducer, in this case IPTG, which turns on the excision machinery. Our shuttle construct undergoes site specific recombination to return to it's plasmid state. Pay close attention to the inducible promoter in the modular region at this point.

We can see that physically, our constructs are in the same state that they were initially. However, at this point, the chemical inducer is present. This means that the pir gene and the CI repressor of the shuttle construct are expressed. The product of the pir gene replicates the shuttle construct to a high copy number. The C1 repressor prevents the reintegration of our construct by down regulating the integrase machinery. Our excision machinery was turned off due to the excision event itself. We have now successfully integrated and retrieved the file. Upon removal of the inducer, we should be able to repeat this process.

One potential problem we wish to look into furthur, is reintegration in the presence of a high copy number plasmid.