From 2014.igem.org

Latest revision as of 15:23, 17 October 2014

Summary

The most refined method of transforming Marchantia is through A. tumefaciens mediated transformation. This is done using a binary vector: a Ti ("tumor inducing") plasmid and a T-DNA plasmid. The Ti plasmid contains the parts to enable agrobacterium to transfect Marchantia, and the T-DNA plasmid contains the DNA to be transfected.

It is recommended to prepare a stock of electrocompetent agro with the Ti plasmid, so when creating constructs, you only need to change the the T-DNA plasmid.

The T-DNA plasmid usually looks as follows:

The T-DNA plasmid contains the 35S promoter and nosT terminator flanking the gene of interest

The region in between the Left Border (LB) and Right Border (RB) is the T-DNA (Transfer-DNA), which is into the MP genome. The rest is to be able to replicate the plasmid in AT and E.coli, and is usually constant between construct to construct.

The T-DNA is the region which you need to design and assemble. Usually, since we want to select transformants, we use the selection cassette (BBa_K1484316). It is usually placed adjacent to the LB, since this is the last point to get integrated into MP, so transformants with only partial integration of the T-DNA will not be selected.

In between the LB and RB, genes must follow the format:
promoter - 5' UTR - coding sequence - 3' UTR - terminator
You should assemble your constructs following this pattern. Parts function differently between Marchantia and other plant chassis. See appropriate sections of this wiki for more information about these.

For the most basic Marchantia construct expressing a given coding sequence, follow the plasmid map above. That is, replace GOI with your coding sequence.

Note: if you are using MoClo, level 1 acceptor vectors are ready to be electroporated into AT. You don't need to worry about assembling your construct into the appropriate T-DNA vector.