Developing a frugal transcription factor relative affinity measurement pipeline (TRAMP)

 

Our goal is to develop a new method that will facilitate the design of synthetic promoters with varying strength or specificity and will be a useful tool for genome engineering.

The Idea

Plant synthetic biology applications require the availability of a versatile set of promoters with different strength or specificity. Currently the number of promoters for plant synthetic biology is extremely limited. Our goal is to develop a new method that will facilitate the design of synthetic promoters with varying strength or specificity and will be a useful tool for genome engineering.

It has been shown that both the activity strength and the specificity of a promoter are heavily influenced by the affinity of a transcription factor (TF) to its binding site (TFBS) (Farley et al. 2015; Farley et al. 2016). Techniques to assess the affinity of a TF to its TFBS, such as the protein binding microarray (PBM), are useful tools for identifying novel TFBS and comparing TFs binding affinities (Berger & Bulyk 2006). However, such techniques are still not widely affordable. Microplate-based protein-DNA binding assays have been developed as a cheaper alternative, with the only limiting factor being the cost of the necessary DNA modifications.

In this project, we aim to establish a frugal platform for comparing relative TF-TFBS binding affinities by utilising a non-modified DNA immobilising method. This method is based on the commercial DNA coating solution (Thermo and Abcam) which has been used in the chemiluminescence immunoassay to assess DNA adducts levels (van Gijssel et al. 2002).  We plan to proceed in two main stages:

1) Setting up a transcription factor relative affinity measurement pipeline (TRAMP) and optimising its parameters.

2) Demonstration of the usage of TRAMP by applying it to a practical project designing a set of promoters with different activity strengths and function in different species.

The Team

Dr Yaomin Cai,
Postdoctoral researcher, Engineering Biology, Earlham Institute

Dr Will Nash,
Postdoctoral researcher, Bioinformatician/Evolution biology, Earlham Institute

Dr Susana Sauret-Gueto,
OpenPlant Research Manager, Prof Haseloff Lab, Plant Sciences Department, University of Cambridge

Dr Eftychios Frangedakis,
Research Associate, Prof Haseloff Lab, Plant Sciences Department, University of Cambridge


Project Outputs

Project Report

Summary of the project's achievements and future plans

Project Proposal

Figure 1; Figure 2; Figure 3

Original proposal and application

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Project Resources


Jim Haseloff