Team:WashU/YLCExperiment

Experiment

Introduction and Design

In order to give this demonstration at the Youth Learning Center (YLC), we are creating the necessary materials ahead of time. The development of 5 parallel constructs (eYFP, GFP, mRFP1, eCFP, and mCherry) all with the same ribosome binding site (RBS) and set of double terminators was our challenge. (Initially, two YFP constructs were considered with different RBS's, but no primers for these were ordered.) We wanted to create parallel constructs so that the only differences in expression levels and colors would be the open reading frame's protein and not the promoter or RBS efficiency. In a sense, we want to develop a simple BioPaint Set that we could let the students at the YLC could view as a realistic application of genetic engineering.

The decision to create a BioPaint set was developed after meeting with some of our advisors and discussing our options. We developed several ideas including colors with different resistances, different plasmid backbones, and different promoters (constitutive versus induced versus inhibited). Ultimately, the choice to make a BioPaint Set came down to feasibility and ability to characterize previous BioBricks well as this was not our premier project of the summer. This project easily lent itself to these goals and was thus selected for production.

We began our research into the parts. Although many BioBricks existed, we wanted to build our own so that we could control the differences and create constitutive color with the greatest expression possible. As a result, we decided to use previous BioBricks to create our products. J23100, a sigma-70 constitutive promoter in E. coli, was initially selected as the promoter of choice. The five fluorescent proteins were selected and can be found on our parts page. These were selected for their parallel constructs, engineered coding regions, and convenience of their presence on the initial iGEM plates.

Developing the Constructs

Initially, the different DNA plasmids from the wells on the plates were hydrated with autoclaved dH2O and cloned into Sigma-Aldrich GC5 competent cells using the general transformation protocol. Since all the parts were Ampicillin-resistant plasmids, the transformed E. coli were plated onto LB-Amp plates.