Introduction

These are our list of results that we have successfully acquired through our two-year run developing this project. Special thanks to Lisa Oberding for helping with our Gene Design, Gene Synthesis, and Transformation.

Gene Design

The 4 constructs that we have designed, with the help of our mentor Lisa Oberding and previous iGem teams (especially Tianjin 2016), include:

• a polyethylene terephthalate hydrolase (PET-ase) fused to a red fluorescent protein, (or RFP) called mCherry, which gives the protein its colour aspect,
• a hydrophobin called BslA fused to mCherry,
• a PET-ase without the RFP, and
• a BslA without RFP.

Special care was taken to ensure the sequences did not include any extra restriction enzyme sites and to design the coding regions to be expressed successfully in BOTH E. coli and B. subtilis cells. See our parts pages for further details regarding these parts.

Implemented prototype, what could be seen in a sorting facility.

Gene Synthesis

The gene constructs we have created with the help of Lisa were synthesized by Bio Basic and shipped to us for transformation. The plasmids were ordered both in the standard pSB1C3 backbone, as well as a second “shuttle” backbone we hope to use in the future for testing on Bacillus specifically.

Transformations

Our lab tried repeatedly to transform our cells. We had success with E. coli but were unable to transform Bacillus subtilis on our own. Thank you to Amino Labs who hosted us over the summer for a second (though unsuccessful) round of attempts at Bacillus transformation. Thankfully our mentor, Lisa Oberding from Fredsense Technologies, was able to help us successfully transform both E.coli and B.subtilis cells with each of our four constructs.

These pictures are our constructs in E.coli DH5alpha. The ones on the left are imaged under regular light and the picture on the right is under UV light. The coding system Lisa used for the test is:

• E - PET-ase
• F - mCherry PET-ase
• G - BslA
• H - mCherry BslA

Regular (left), UV (right)