Comparative analysis of cell free and in planta protein synthesis systems

Our aim is to optimise a high-throughput protein synthesis method primarily for wheat transcription factors (TFs)

The Idea

This project brings together expertise from the Earlham Institute (Hall and Patron Labs), John Innes (Philippa Borrill) and the Hibberd Lab in University of Cambridge (Pallavi Singh). Our aim is to optimise a high-throughput protein synthesis method primarily for wheat transcription factors (TFs). We propose using a high throughput Golden Gate cloning strategy to create constructs that will allow us to directly compare yield from cell free and in planta protein synthesis systems. This funding would foster collaborations between groups from synergistic areas of plant biology, provide useful data for the synbio community and support future transcriptional network research in wheat.

Wheat is a primary world food crop. It’s production needs to increase to meet demand and understanding regulatory networks will be critical for designing crops for the future. Although we have good understanding of key networks in the model plant Arabidopsis, the extent to which they have been conserved in wheat remains an open question. In addition, we have very little understanding how gene regulatory networks operate across complex polypoid genomes. Phylogenetic analyses have revealed many core network transcription factor (TF) homologues, but knowledge of sequence specific binding sites and downstream targets will be required to determine the extent to which they can be considered functional orthologues. DNA Affinity Purification Sequencing (DAP-Seq) is a high throughput method that can provide both of these key pieces of information (O’Malley et al., 2016). It uses epitope tagged TFs to pull out associated DNA sequences to reveal binding sites. A recently published DAP-seq dataset for Arabidopsis transcription factors has paved the way for similar studies to be carried out in other plant species and Susan is leading a project in the Hall lab to set this up for wheat. We are requesting Open Plant funding to extend the optimisation of protein synthesis section of this technique with the aim of improving the ~30% DAP-Seq success rate reported for Arabidopsis TFs. It will allow us to extend the scope of the project and carry out a comprehensive test of three protein synthesis methods. We propose comparing cell free and in planta synthesis systems for 42 wheat TFs which include homologues of key Arabidopsis TFs involved in circadian rhythms and photosynthesis as well as a characterized TF that is known to be involved in wheat senescence.

The Team

Dr Susan Duncan,
Postdoctoral Researcher, Organisms and Ecosystems Department, Earlham Institute, Norwich

Dr Laura-Jayne Gardiner,
Postdoctoral Researcher, Organisms and Ecosystems Department, Earlham Institute, Norwich

Dr Quentin Dudley,
Postdoctoral Researcher, Engineering Biology Department, Earlham Institute, Norwich

Dr Philippa Borrill,
Research Fellow, Department of Crop Genetics, John Innes Centre

Dr Pallavi Singh,
Postdoctoral Researcher, Department of Plant Sciences, University of Cambridge


Project Outputs

Project Report

This project is due to report in 2018.

Project Proposal

Original proposal and application

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Project Resources