• Research

Protein synthesis: in and out of cells

Comparative analysis of cell free and in planta protein synthesis systems

Project summary.

Led by: Anthony Hall Group

Start date: January 2018

End date: December 2018

Duration: 1 year

Grants: OpenPlant

Value: £5,000

Wheat is a primary world food crop which provides calories to more people than any other, meaning that in order to feed our rapidly expanding global population we must be able to increase our yields year on year by around 1.6%.

Understanding gene regulatory networks will be critical for designing crops for the future in order to meet this sort of demand. 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 of how gene regulatory networks operate across complex polyploid genomes.

This project aims to tackle both of these questions using DNA Affinity Purification Sequencing (DAP-Seq) in both cell free and in planta protein expression systems to generate tagged transcription factors that can reveal associated DNA sequences and binding sites.

Details.

The project aims to compare cell free and in planta synthesis systems for 42 wheat transcription factors (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 project is split into four objectives as follows:

  1. Synthesis of 42 freely available DNA modules for wheat transcription factors
  2. Valuable comparative data for in vitro cell free and in planta protein synthesis methods
  3. An optimised protein synthesis pipeline that will underpin an ongoing large DAP-Seq project
  4. New collaborations that would promote knowledge exchange and capitalise on expertise across EI, JIC and the University of Cambridge

Thanks to the success so far of the project, we now have an economical in-house E. coli cell free protein synthesis (CFPS) expression system based at Earlham Institute, which has been used extensively and now has a proven success rate for wheat TF synthesis.

Tools.

Cell free protein synthesis (CFPS) at the Earlham Institute.

Collaborators.

Philippa Borrill, John Innes Centre

Pallavi Singh, University of Cambridge

Impact statement.

OpenPlant funding has provided us with a strong foundation from which to pursue our goal - to generate the first genome-wide TF binding site dataset for wheat.

Thanks to the success so far of the project, we now have an economical in-house E. coli cell free protein synthesis (CFPS) expression system based at Earlham Institute, which has been used extensively and now has a proven success rate for wheat TF synthesis.

Related reading.