Project Design

Step 1: Produce Cassettes

Gene Cassettes are the carriers of genetic information in our design. They are small, modular chunks of DNA containing a gene flanked by AttC sites. In their mobile circular form, cassettes, unlike plasmids, are non-replicative and so do not require extra genes to modulate or initiate replication. To create a cassette simply requires the use of primers to create half AttC sites at either end and circularizing the product by using Enzymatic Ligation Assisted by Nucleases (ELAN). This is a reaction in which ligation and enzymatic digestion of compatible but non-identical ends, like XbaI and SpeI, occurs simultaneously. This should result only in stable single-cassette circles or concatemers of several cassettes. For systems that already contain an intact AttC site, XbaI and SpeI sites can be introduced by PCR for ELAN, or overlapping ends can be introduced by PCR for Gibson Assembly.

Step2: Express Integrase

As gene cassettes are non-replicative it is important that they are incorporated into an AttI site before the cell divides and the cassettes face the possibility of ejection. Therefore it is vital to have a controllable Integrase gene which can be turned on prior to the addition of gene cassettes to ensure that the cassettes are integrated before they are lost. Part pUS203 is our submission BioBrick which encodes the controllable Integrase system. We are using the araC-pBAD system from K731201 which allows us to turn on transcription of the Integrase under the activation of arabinose.

By controlling integrase expression with arabinose, we are also able to turn off integrase, which would stop inappropriate excision of cassettes from the array from occurring after the array has been assembled.

Step 3: Add Cassettes

As gene cassettes enter the cell site the integrase acts to cause specific recombination between the AttC site on the cassette and an AttI site which may be present on the plasmid or bacterial chromosome. Integrase facilitates the formation of a holiday junction between the AttI and AttC regions at the recombination site, resulting in insertion of the cassette into the middle of the AttI site.

Step 4: Express the array

Once the cassette is integrated, we would induce expression of the genes using an inducible promoter which is placed upstream to the AttI site and cassette array. A single promoter can read through multiple cassettes if a terminator is not present, allowing multiple genes to be expressed in one go.

With thanks to: