Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

Matthew D. Mattozzi; Mathias J. Voges; Pamela A. Silver; Jeffrey C. Way

doi:10.3791/51234

A subscription to JoVE is required to view this content. Sign in or start your free trial.

Method Article

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

20.4K views

⸱

DOI:

10.3791/51234

⸱

April 18th, 2014

Matthew D. Mattozzi¹^,² , Mathias J. Voges¹^,²^,³ , Pamela A. Silver¹^,² , Jeffrey C. Way¹^,²

¹Synthetic Biology Platform, Wyss Institute for Biologically Inspired Engineering, Harvard University, ²Department of Systems Biology, Harvard Medical School, ³Department of Biotechnology, Delft University of Technology

Summary

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

Abstract

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5’ mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work¹¹, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

Introduction

This work is a metabolic engineering / synthetic biology project wherein plant cells are engineered to express a reporter protein in multiple organelles but with only a single DNA construct.

One approach to target proteins to more than one location involves cloning multiple genetic copies, each containing a different localization peptide. Each copy must be introduced by successive retransformation, or alternatively, by backcrossing single transforms¹. This involves additional cloning, and is limited by one localization tag per terminus.

Another way to localize a protein to multiple locations is through....