Hosted by Thomas Gorochowski, School of Biological Sciences

It is a well known problem in engineering biology, that our habit of fluorescent protein (FP) quantification in relative units inhibits our ability to connect circuit characterisation data with mathematical models. Such practice obscures even basic information, such as the order of magnitude at which engineered cells produce proteins, as well as the relative levels of multiple FPs within the same cell.

We have previously demonstrated that it is possible to extract absolute copy number information from fluorescence measurements using FPs as calibrants (using a method we named FPCountR), ie. that an mCherry calibrant can be used to extract mCherry copy numbers in a plate reader experiment. In recent work, we expand FPCountR by demonstrating that spectral correction allows the use of independent calibrants to do the same, ie., that mCherry calibrants can be used to quantify all red FPs, or that mRFP1 or sulforhodamine calibrants can be used to quantify mCherry. We validate this method by testing a wide range of small molecule fluorophores for their potential as calibrants (a range of the brightest commercially available dyes including the iGEM calibrants) and discover that some perform poorly due to environmental sensitivity, while others are more robust. The resultant method simplifies calibration to a fraction of the time and cost of previous methods, enabling easy access to biologically meaningful units for engineering biology.

To expand our investigation of the relationship between molecular copy numbers and circuit performance, we are also interested in counting ribosomes in living cells via the insertion of fluorescent RNA aptamers into ribosomal RNA. We have identified multiple insertion sites within the 16S and 23S ribosomal RNAs with minimal effect on translation activity, allowing dynamic ribosome quantification in living cells. While absolute RNA counting in living cells is currently challenging, recent developments work towards building methods that enable this in the near future.

Lab website: https://www.csibra-lab.com/