Characterization of the Performance of Non-Fluorescent Genetic Constructs via Indirect

Competitive Construct Characterization (iCCC)

Ariel Hecht, Matthew S. Munson, Drew Endy, and Marc Salit

Efficient engineering of complex metabolic pathways into organisms depends on the ability to predict the function of a composed system from knowledge of its constitutive parts. Our work is focused on the development of methods for parts characterization that yield reproducible results that can be meaningfully compared across different experiments, different labs, and different times. This capability could yield a database of engineering reference data that can be used with confidence to support design, reusability, and predictive modeling.
Current methods for characterizing circuit performance require including a fluorescent protein gene in the construct of interest, which can significantly alter its behavior.1,2 To overcome this limitation, we are developing the indirect Competitive Construct Characterization (iCCC) assay as a method for quantifying the performance of any non-fluorescent genetic construct.
We propose an assay system where the construct of interest is expressed in competition with a reference construct producing a fluorescent protein in a resource-limited environment. The use of a cell free expression system allows for engineering control over the resources available for transcription and translation, allowing the environment to be tailored to ensure competition between the constructs. The ratio of fluorescent protein produced under competitive and non- competitive conditions, should be a robust measure of the activity of the part of interest. The key assumptions underlying this method are:
(1) Consumption of resources by a construct is a meaningful measure of its activity
(2) Resource limitations affect the activity of both constructs in a known manner
(3) In a resource limited environment, consumption of resources by the construct of interest results in reduction of protein production by the reference construct
We have adapted and applied mathematical models3â??5 of transcription and translation of two constructs coupled through their utilization of shared resources (NTPs, amino acids, polymerases, and ribosomes). We will show the use of this model to explore parameter space allowing determination of regimes under which competition for resources can be most meaningfully observed as a decrease in the production of the reference construct. We use this to provide definition of the potential operating limits of the assay, as well as guide design of the characteristics of useful reference constructs.

References

1. Mutalik, V. K. et al. Precise and reliable gene expression via standard transcription and translation initiation

elements. Nat. Methods 10, 354â??60 (2013).

2. Mutalik, V. K. et al. Quantitative estimation of activity and quality for collections of functional genetic elements. Nat. Methods 10, 347â??53 (2013).

3. Siegal-gaskins, D., Noireaux, V. & Murray, R. M. Biomolecular resource utilization in elementary cell-free gene circuits. Am. Control Conf. (ACC), 2013 1531â??1536 (2013).

4. Stögbauer, T., Windhager, L., Zimmer, R. & Rädler, J. O. Experiment and mathematical modeling of gene expression dynamics in a cell-free system. Integr. Biol. 4, 494â??501 (2012).

5. Niederholtmeyer, H., Stepanova, V. & Maerkl, S. J. Implementation of cell-free biological networks at steady state. Proc. Natl. Acad. Sci. U. S. A. 110, 15985â??90 (2013).