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Revision as of 20:45, 20 September 2012

Plasmids

Xenopus tropicalis representes a challenge as it is not only a vertebrate, but also a new chassis in the iGEM competition. To make sure we meet iGEM’s expectations on time, we have had to develop a new biobrick plasmid backbone compatible with Xenopus tropicalis.
This plasmid backbone for eukaryotes contains:
• RBS (Kozac)
• Promoter
• 5' and 3’UTR for 5' mRNA capping and 3'
• PolyA tag
• Antibiotic resistance genes
• Origins of replication
• Biobrick cloning site
By putting all the parts necessary for expression in eukaryotes, we have made plasmids where any biobrick can be cloned in directly without having to transfert each parts individually. These plasmids can be used to rapidly test genetic constructs in Xenopus after a single cloning. The plasmid also contains tools to calibrate the system in combination with a model. For instance, it contains sites for in vitro transcription of genes to make RNA that can then be injected directly in the embryo, allowing a much finer control of the ratio between levels of different genes during construct testing. The way we test it in Xenopus is by injecting a high concentration of plasmids straight into the embryo at the first stage when there is only one cell. As the cell divides, the plasmids are shared between cells but not replicated so a high concentration of DNA is necessary to ensure there will be DNA in most of the organism. Once the transcription machinery turns on during the development, plasmids are transcribed and translated. Two weeks atfer injection of plamids containing GFP, we could still see the expression of GFP throughout the embryo. Since the tadpole stage starts after a few days, we can work on a whole vertebrate with most organs formed within a week. With different tissues it is possible to diversify the type of expression with different promoters. So far we have made 3 new plasmid backbones with different promoters so we can choose to test them in a ubiquitous (CMV promoter), inducible (Hsp70 promoter) or a tissue specific manner (Elastase promoter). Our goal was to provide the tools which would allow to rapidly build and characterize constructs in the embryos (along with a synthetic hormone to make them communicate), with the tools for debugging (by injecting mRNA) and with ubiquitous, inducible and tissue specific promoters. Once the system is tested using the injection method, the final parts can then be made using traditional biobricks and inserted onto the Xenopus chromosome - but this requires experiments on a much higher number of embryos, with more complicated protocols and lower chances of success.