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Team:USyd-Australia/Project/Approach
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Approach
E. coli does not have a naturally occuring integron system so it lacks an AttI site and also does not express the integron Integrase. For our system to work we need to introduce these components into our cell which will then allow cassettes to be integrated.
The choice for an AttI site in our project came from what was available and often used in the lab. The AttI site used was on a plasmid with Streptomycin resistance (referrenced as pUS44) which fit well with the rest of our experimental set up.
The design of the controllable integron integrase was based of the Paris-Bettencourt 2010 araC-pBAD Integrase BioBrick. We started with araC-pBAD in pSB1C3 which had been previously prepared by an Honours student in the lab, Sam Ross, from araC-pBAD provided in the iGEM kit. The integrase gene was was ordered as a gBlock with the sequence for that coming from pUS2056. The Integrase gene is highly conserved though so this choice was mainly out of convenience. The gBlock was inserted into the SamR construct to give us a controllable Integrase system in a proper format for submission.
While the integrase was in a controllable system it is believed that a high concentration of integrase is toxic to the bacterial cell. As such we also needed a low-copy chassis into which we could insert the controllable Integrase system created previously. We decided that it would be best to improve on the pSB6A1 low-copy plasmid provided by iGEM by adding in the BioBrick prefix/suffix and terminators from pSB1C3. This part would then have been inserted with the controllable Integrase system and would have allowed us to carry out experiments for the activity of the modified Integrase gene.
The final component needed was a gene cassette. The template for the cassette was a gBlock based of the sequences of aeBlue-aacC1 GmR and half AttC sites on either end. A PCR of the whole template gave a linearized cassette and an ELAN reaction circularized the cassette, giving a full gene cassette with colour and selective markers for antibiotic resistance.
Our initial plan was to create and validate these parts separately and test their expression together.
