Seeing the Invisible: Exploting Crosstalk to Measure Genetic Constructs In Vivo

The emerging bioeconomy is in need of a metrology infrastructure. One area where infrastructure is needed is for the measurement of the activity of genetic constructs that do not produce fluorescent proteins. This abstract describes a competitive assay for measuring genetic construct activity in Escherichia coli.

In cells, transcriptional and translational resources are limited, resulting in a limited capacity for protein expression. It has been shown that genetic constructs compete for resources, with expression of one construct coming at the expense of other constructs. Our assay leverages this competition to measure the activity of spectroscopically invisible genetic constructs. Several new results from previously presented work are highlighted here:

1)    In vivo there is a monotonic relationship between the co-expression of two constructs, where increases in expression of one lead to decreases in express of the other.

2)    We exploit this relationship to develop a calibration method where we can infer the expression level of an undetectable protein, by observing the reduction in expression of a fluorescent reference protein.  

3)    We show the impact of reference construct copy number and expression strain on the performance of the assay.

These data show that the competitive assay can be used to measure the expression level of spectroscopically undetectable constructs, and can be useful for enabling forward engineering in synthetic biology.