Introduction

As we were planning our project we considered the yellow fluorescence protein mVenus to be a straight-forward screening tool. With our first successful transformations we realised how wrong we were. Equipping an organism full of pigments and photoreceptors like C. reinhardtii with a fluorescent protein for concentration measurements is quite brave, but using it for screening seemed to us to be a manageable task. Our first plate reader results taught us otherwise. With every measurement indicating a higher fluorescence for wild type algae then for the transformed, frustration started to rise. Measuring YFP in C. reinhardtii turned out to be like looking for a fluorescent needle in a colorful haystack.

Our first approach was to perform colony PCRs on every YFP-construct colonies that were picked, something we were trying to avoid. We had hoped to be able to use YFP as a fast screening tool for our successful transformations. Every clone that showed a positive band in the PCR gel was then observed under a fluorescent microscope. At this point we were able to identify our first YFP clones and that the part worked in principle. Returning to the plate reader our now confirmed YFP clones still showed smaller or equal emission values than our wt strain UVM 4 under the plate reader. One thing we did achieve: more frustration. Only after many measurements and trial and error we were able to identify the correct parameters to measure our YFP. We are aware that for many experts in the field of fluorescence measurement this kind of measurements are part of the daily routine. Nevertheless, our goal is to expose our mistakes and troubleshooting when measuring YFP in C. reinhardtii to help other young researchers and iGEM teams that might find themselves in a similar situation.