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From 2012.igem.org

Xenopus tropicalis: A new multicellular chassis

Establishment of a new chassis

So far, synthetic biology has mostly focused on bacteria, since they are simple to engineer. iGEM teams and laboratories have worked on unicellular organisms in order to understand the underlying biology and have developed an impressive database of molecular parts. Some work has also been done on engineering mammalian cells and a few iGEM teams have followed this trend. Synthetic biologists are now imagining the rational design of multicellular organisms with numerous applications ranging from gene therapy or drug production to environmental monitoring. This year, our team would like to be part of that challenge.

The arrival of Xenopus as a chassis in synthetic biology requires the creation of new standards and protocols that the community will be able to build on. We provided the registry with such tools that allow rapid construction and characterization of devices in vivo, and include debugging tools. We think they will be very useful for later iGEM teams and synthetic biologists who wish to work with Xenopus for building multicellular systems.

This year, the Evry iGEM team is going to be the one of the first iGEM team to work on a vertebrate. Our work is focused both on developing a system for intercellular and intertissue communication, and creating the tools for the iGEM community to easily express genes in specific tissues. We believe the tadpole is a chassis of choice for iGEM on multicellular organisms, as experiments can be conducted in one week using microinjection methods. We hope to demonstrate the feasibility of engineering Xenopus in one summer for an iGEM project, and to create a great tool for multicellular synthetic biology: An orthogonal hormonal system.

The simple molecular strategy to build eukaryotic plasmid ready to use:

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 (but also others vertebrate and fish).

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. The promoter is surrounded by the enzyme restriction sites SalI and HindIII in order to be able to switch more easily to other promoters,

5'UTR and 3’UTR : UTRs are Untranslated Region. The B-globin 5' UTR is located upstream of the coding sequence and is involved in 5'RNA capping. 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,

SV40 PolyA signal : The simian virus 40 polyadenylation signal 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,

Origin 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.

Example of GFP expression in Xenopus

The injection tutorial explains very simply with diagram how we did injection and how take care about your embryos and tadpole. The experiment carries on 5 days, from the unfertilized egg to a swimming tadpole at stage 48-50. The GFP (or other fluorescent protein) is expressed few hours after the fertilization to the end of the week (see below).

Plasmid injected: pCS2+ with CMV promoter and GFP-aid reporter

pCS2+ GFP-aid: this plasmid contains the constitutive and ubiquitous promoter CMV and the aid sequenced of the aid system fusionned to GFP (Green Fluorescent Protein)(Nishimura et al., 2009), this Biobrick created by our team is BBa_K812010, and it was integrated into our Eucaryotic plasmid BBa_K812000.We injected about 3.78E+7 plasmids.

GFP-aid expression from the embryo at stage 20 (one day after injection )to a tadpole at stage ~46 (four days after injection). For this tadpole the expression is localized in the skin.

The characterization of all reporter and promoters is here.

Conclusion

We showed that our constructions express our reporters with the CMV promoter. These parts are considered characterized. Nevertheless we expected a more uniform expression of the reporters with the CMV promoter. Spectral variants of fluorescent proteins could be expressed in different tissues. Within one tadpole the fluorescent proteins were observed in one to four different tissues, and tissues were different between tadpoles.

Explanation: The plasmid DNA does not diffuse in the egg and stay in the same area around the injection site. This means that depending on the injection site, the plasmid will be inherited by a given set of cells within the tadpole. This question was raised in our model. Another reason could be that the metabolism of each differentiated cell is different and changes during the tadpole's development.

Moreover the expression of reporters decreases during time, because plasmid DNA is subjected to a catabolic activity during development but also plasmid DNA gets diluted as cells proliferate and the quantity of plasmid DNA decreases for each cell.