Team:Cambridge-JIC/Homoplasmy
HOMOPLASMY STRATEGY
A video has been prepared below to help explain our homoplasmy strategy. A transcript of the video can also be found below
Transcript
The Cambridge-JIC 2016 IGEM team has developed a chloroplast transformation toolkit to allow future teams to use plastids as a new chassis to implement genetic circuits and exploit the potential of plastid engineering.
Chloroplasts hold a massive potential as an alternative protein expression system, due to their outstanding expression yields, diversity of post-translational modifications and auto/mixotrophic lifestyles of plants and microalgae. They are also the target of research, aiming to enhance crop and biofuel yields.Various proteins have already been successfully expressed in chloroplasts, including monoclonal antibodies , antigens, anti-toxins and growth factors.
Unfortunately, chloroplasts engineering has not been fully explored, due to a series of bottlenecks, which we tried to tackle with our project. One of them is that homoplasmy, the last step of chloroplast transformation leading to the stable integration of the transgene in the genome, takes 2-3 months to achieve. We have developed a strategy which may, in principle, significantly decrease this time. This strategy was designed specifically to be used in model organisms with single chloroplasts, such as the microalgae Chlamydomonas reinhardtii, and may not be applicable to higher plants.
Homoplasmy is a term used to describe a cell whose plastid genome copies are identical, or in our case, have all been transformed. After initially transformation, only a few our of over 80 copies of the chloroplast genome take up the transfected cassette. It then usually takes months of segregation driven by antibiotic resistance selection pressure, to achieve homoplasmic strains.
Our team has designed a system in which this can be achieved much quicker with CRISPR/CAS9 technology. This video will explain our design. Additionally, you can find we have generated a parts library as well as hardware and modelling to make the necessary tools to implement this idea.
Our team has designed a system to accelerate the spread of any cassette of interest among the genome copies with the use of CRISPR/Cas9 technology. It depends on a co-transformation of the chloroplasts with two plasmids, the “driver” and the cassette-of-interest. The device is divided into two separate plasmids to ensure the the biological containment of the cas9 protein, as discussed later.
Designing for the DIY Bio Community
In order to best understand the requirements for our hardware, we contacted community labs from around the world to inform them about our project and find out the features they wanted to see in our designs.
We contacted the labs listed in the table below, asking about their current experience with plant synthetic biology, existing laboratory resources and technical experience in building hardware. This was to ensure we were creating accessible designs, both financially and in their technical demands for construction, that would definitely fulfill a real need for these groups.
The full survey and information about our project sent to these labs can be seen here.
| Organisation | Contact | Feedback/Comments | Comments integrated into project design |
|---|---|---|---|
| The Lab, LA's Hub for Citizen Science and DIYBio | Cory Tobin | Wide range of technical skills in their hackspace and interest in DIY bio hardware, but strong budget constraints on building devices. Full survey response download |
Ensuring low cost of designs and thorough documentation of all the parts used |
| London BioHackspace< | Tom Hodder | Previous experience of building open source designs, such as openPump syringe, and strong interest in building our designs. Full survey response download |
Ensuring low cost of designs (below 500 GDP specifically for the gene gun), and consumables required for these |
| bioCURIOUS, Silicon Valley | Jay Hanson | Previously built their own DIY gene gun and have published this at their website | Need for achieving pressure >100 psi in order to penetrate plant cells (from their own testing experience) |
| Bioflux Society, Berlin | Alessandro Volpato | Interest in receiving our design and lab protocols, and in working with microalgae | Integrating more safety features into the design, and checking these with thorough pressure testing |
