From 2012.igem.org

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:

[[File:image-1_PR.png|400px]]



RBS (Kozac) : The Kozak consensus sequence is essential for the initiation of the translation process in eukaryotes. The sequence is the following (gcc)gccRccAUGG, where upper case letters are highly-conserved bases, and lower case letter can vary,
Promoter : The promoter of a gene is located upstream of this gene and initiate its transcription
5'UTR and 3’UTR : UTRs are Untranslated Region. The 5' UTR is located upstream of the coding sequence and may contain elements for controlling gene expression. The 3'UTR is located downstream of the coding sequence and may contain sequences for the regulation of translation efficiency, mRNA stability, and polyadenylation signals
PolyA : This sequence is involved in the maturation of the mRNA for translation and is composed of a succession of adenine bases
Antibiotic resistance genes : This sequence is necessary for the selection of transformed bacteria exposed to the antibiotic,
Origins of replication : Sequence initiating the replication of DNA,
Biobrick prefix and suffix : The BioBrick prefix contains the restriction sites for EcoRI and XbaI and the BioBrick suffix for SpeI and PstI.


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 tropicalis 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 tropicalis is by injecting 2.3 nL of plasmids at 100 ng.uL-1 straight into the embryo at the first stage when there is only one cell. For a plasmid of 4kb it represents approximately 45 million of plasmids per injection. 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. 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.
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