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From 2014.igem.org

Approach

Introduction

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.

AttI Site

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.

Controllable Integrase expression

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. This plasmid is referenced to as pUS203 throughout the wiki.

Low-Copy Backbone

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 low copy plasmid is referenced to as pUS201 throughout the wiki. This part would then have been inserted with the controllable Integrase system from pUS203 and would have allowed us to carry out experiments for the activity of the modified Integrase gene.

Gene Cassette

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. This cassette was inserted into the pSC1B3 backbone and this plasmid is reference to as pUS204 throughout the wiki.

Experimental plan

Our initial plan was to create and validate these parts separately and test their expression together. Head over to the Results and Conclusions page for outcomes of these components and our project.

With thanks to: