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Team:NRP-UEA-Norwich/Project System
From 2014.igem.org
Our System
We have chosen to work with not only bacterial but also a plant chassis!
Escherichia coli
As all BioBricks submitted to the iGEM registry are designed to work in the model organism Escherichia coli we utilised this microorganism to allow us to clone our gene constructs successfully, ensuring our parts would be available for use by future iGEM teams. E. coli proved useful as a chassis as it has a rapid doubling time of less than an hour, this allowed cultures to be saturated O/N and a mini-prep and diagnostic gel run the following day, allowing for swift transformation into our second chassis Agrobacterium tumefaciens. The E. coli K12 strain which we utilised to clone our constructs is a biosafety level 1 organism, therefore poses minimal threat as cannot thrive in the gut.
Agrobacterium tumefaciens
A. Tumefaciens proved vital as a chassis organism in this project as acted as a shuttle chassis for the delivery of our Level 1 and Level 2 DNA constructs, once cloned in E. coli and DNA purified into the plant species and our final biosensor organism Nicotiana benthamiana. Agrobacterium tumefaciens infects plants through its Ti plasmid. The Ti plasmid inserts a segment of its DNA, termed T-DNA, into the host organisms genome when the T-DNA is successfully integrated this causes the expression of the gene of interest by the plant. The T-DNA can be altered to include any gene construct that you would like, so it can be inserted into the host cells. Below are three cartoons showing a simplified version of Agrobacterium tumerfaciens transfection.
Figure 1. The Agrobacterium tumerfaciens cell attaches to the plant cell and the VirA and VirG proteins sense the plant hormones (PH). The T-DNA in the T-region (red) of the Ti plasmid can be excised upon activation of the Vir region (yellow) by the VirG protein.
Figure 2. Upon activation of the VirA protein by the plant hormones (PH) the VirG protein dissociates and activates the Vir region (yellow) and causes the excision of the T-DNA. The Agrobacterium tumerfaciens also utilises a type IV secretion system (channel) through which the T-DNA (coated with VirD2 and VirE2 proteins) can transfer into the plant cell and are localised to the nucleus.
Figure 3. The T-DNA localises to the nucleus of the plant cell where the VirD2 proteins interact with the nuclear pore complexes (NPCs, orange) and allow the T-DNA to enter the nucleus. The VirE2 proteins protect the DNA from being recognised by the host (plant) and degraded. Once inside the nucleus the VirD2 and VirE2 proteins are stripped from the T-DNA and the DNA can be integrated into the genome, or transiently expressed as was the case with our system.
Nicotiana benthamiana
N. benthamiana is a widely used experimental plant from the solanaceous group of flowering plants that includes tomatoes, potatoes and capsicums. It is widely used in plant pathology due to the large number of plant pathogens (viruses, bacteria, fungi, oomycetes etc) that can successfully infect it. Of importance to synthetic biologists, N. benthamiana, is easily genetically transformed, regenerated and amenable to facile methods for virus-induced gene silencing and transient protein expression including the production of therapeutic compounds and pharmaceuticals. We selected it as the plant biosensor as it is a model organism for the plant world.
Figure 4: Nicotiana Benthamiana leaf infiltrated with sterile water to act as a negative control for Agrobacterium tumefacians infiltrations.
