Plant ProChip: Development of a microfluidic device for high-throughput analysis of genetic circuits in plant protoplasts.
Abstract
DNA regulatory elements are fundamental parts for genetic circuit design. The generation and characterization of promoter libraries can greatly facilitate fine tuning of gene expression within a circuit. Current validation techniques involve fusing sequences to a reporter and analysing expression in planta, which requires testing each element in an individual plant, either through transient biolistic transformation or the generation of stable transgenics (Brown et al. 2011). Applying these techniques to a considerable number of genetic circuits can be very laborious or not even feasible at a laboratory scale. The aim of this project was to use microfluidics to develop an inexpensive and high-throughput screening method for the analysis of genetic circuits in plant protoplasts.
Progress
- Generated a fluorescent reporter construct using Golden Gate Cloning.
- Fabricated a PDMS microfluidic device for encapsulation of plant protoplasts.
- Fabricated a PDMS microfluidic device for sorting of plant protoplasts.
- Established of a procedure for protoplast isolation from maize, arabidopsis and Nicotiana benthamiana.
- Established of a procedure for encapsulation of protoplasts.
- Established a procedure for PEG transformation of protoplasts followed by encapsulation.
- Established that protoplasts are stable in a microfluidic device.
- Established of a laser setup for analysis of fluorescence from plant protoplasts.
- Established a procedure for sorting protoplasts based on fluorescence intensity.
Outputs
- A blog which detailed project progress.
- Presentation at OpenPlant Forum, JIC, 2016
- Presentation at Cafe Synthetique, Cambridge, 2016
- Online protocols for protoplast isolation and encapsulation on protocols.io
Plans for follow up
- Develop an on-chip method of protoplast transformation to reduce DNA input requirements.
-
Write up and submission of methods article to an open access journal.