This year, our construct consists of attaching different signal sequences to red flourescent proteins. In addition to E.coli exporting beta-lactamase to the periplasmic space, our project also consists of characterising three different signal sequences:

• K331007 (Beta-lactamase Bla Signal Sequence) that will export the protein to the periplasmic space of the cell
• K331008 (Outer Membrane Protein OmpA) exporting the protein to the outer membrane
• K331009 (Heat Stable Toxin I) which will excrete the protein out of the cell membrane

K331007 relates specifically to our project as it is the signal sequence already present in beta-lactamase. K331009 also relates as it will export our other antibiotic degrading enzymes, such as erythromycin esterase A, out of the cell membrane. We are characterizing these three signal sequences as part of our project this year in order to better understand their functions. Although the Parts Registry outlines where the different signal sequences move the protein to in the cell, it is hard to visually see the protein. In addition we aim to characterize these signal sequences with confocal microscopy to see how effectively they work under different environmental conditions.

Our construct also uses J04500 (IPTG inducible promoter with RBS) as our promoter as it is a well characterized promoter, and was used by the 2012 Lethbridge High School iGEM team for their insulin secretion construct.

To test our construct in the lab, we have used RFP as our gene of interest with a double terminator in order to visually see the export of our protein. When K331007 (beta-lactamase Bla signal sequencing) and K331008 (outer membrane protein OmpA) are used, the protein will be located in the periplasmic membrane and outer membrane of the cell respectively. K331009 (heat stable toxin I) however when used will export the protein out of the cell. Using our dialysis tubing test in the lab, if our construct has K331009 we will see RFP being exported into the surrounding media signalling our construct is successful. From there in our final construct we can replace RFP as our gene of interest with beta-lactamase and erythromycin esterase A.