The aim and rationale
Real biological systems are complicated by the massive number of components and highly complex cellular network. Establishing a cell-like environment in vitro would allow us to disentangle basic mechanisms underlying cellular networks under well-controlled settings. We are a small but interdisciplinary team between the Department of Chemistry and the Department of Plant Sciences, aiming to apply microfluidic droplet system to studying physiological processes in plants, transforming the objects of research from plant tissues into artificial cells.
Microdroplets generation and in-droplet protein expression for mimicking physiological pathways.
The project and more
In this project, we aimed to develop an artificial cellular system using microfluidic droplets that allows the in vitro expression of proteins from plants in a compartmentalised cell-like environment. The biosynthetic pathway of a class of plant pigments, betalains, was used as a simple model to test the methodology, and the TNT® SP6 High-Yield Wheat Germ Protein Expression System from Promega was selected as a reliable in vitro protein expression system. The cell-free protein expression components together with betalain biosynthetic genes were enclosed in microdroplets and intermediate and final products measured.
Betalain biosynthetic pathway from L-Tyrosine to two groups of betalains. DOPA, 3,4 dihydroxyphenylalanine. DODA, 4,5 DOPA dioxygenase. cDOPA5GT, cyclo-DOPA 5-O glucosyltransferase.
Up to this point, we have tested the expression of various proteins in microdroplets. Betalain biosynthetic enzymes were produced and confirmed by fluorescent protein fusions in microdroplets. However, simply co-expressing enzymes from the betalain biosynthetic pathway in the cell-free protein expression solution was not sufficient to drive detectable production of pigment molecules. Following these, optimisation of buffer composition was made and various substrates fed into the system, which led to realisation of parts of the pathway in the in vitro environment.
In addition to the project itself, the various events and training organised by OpenPlant/Biomaker were even more remarkable. The annual OpenPlant Forum gathered an incredibly diverse range of subjects and the low-cost DIY instruments for automating laboratory experiments were mind-blowing.
Future perspective
The OpenPlant/Biomaker project has built solid foundation and connection for us to move forward in the future. Communication between cells plays a central role in many metabolic pathways of multicellular organisms, yet it remains difficult to reproduce in vitro. To this end, an artificial multicellular system with droplet-to-droplet communication will be explored to mimic metabolic pathways which rely on the communication between cells. This platform would open more opportunities for synthetic biology and allow hypotheses and models based on cell-to-cell communications to be tested in vitro.
Last but not least, we thank the OpenPlant/Biomaker teams for the wonderful events and generous help that they have provided!
Written by Zhengao Di, University of Cambridge