From 2014.igem.org

Revision as of 22:44, 16 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.

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.