This is a step-by-step hand holding, descriptive, illustrated picture guide to creating the DNA plasmids that will instruct bacteria how to infect plants and the gene payload to insert into the plant cell’s chromosomal DNA. This guide will not cover the actual stitching together of DNA fragments, and loading it into the bacteria. We’ll be covering tools like UGENE/Benchling and NEB Builder primarily for this.

Overview of Goal

We want to gene edit plants. A common way of doing that is loading DNA on plasmids specialized for agrobacterium, and even more so marchantia.

A common first step for genetic transformations is doing a “reporter” gene, or something that’s easily visible to us. Often people use fluorescent proteins, but we’re going to us RUBY, which will make our plant red/purple! This is wonderful reporter for our plant work, because we don’t need extra tools to see it worked!

• moving parts diagram

Picking Starting Points for Plasmids & Genes

Picking a Plasmid for our Agrobactereum to carry: pMpGWB127

• Plant chromosal DNA needs to be edited. There are many ways to do this, but we’re going to use agrobactereum. Agrobacterum, can do horizontal gene transfer, with a mechanism that’s described as tumor inducing. We’re going to engineer the Ti (tumor inducing) plasmid’s genetic payload to put in the genes we want though: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4292801/.

• Picking a plasmid “backbone” for our agrobactereum

  • A backbone is a shorthand way of saying, the plasmid we’re starting with that contains a lot of functionality we’re just going to add to. In a later step, we’re going to be adding extra genes to turn the plant red. But the functionality of our starting plasmid needs to be able to do the gene transfer.
  • Not all plasmids, not all genes, work the same in all organisms. We’re going to want to use a Ti plasmid specialized for marchantia, called pMpGWB127

    • Plasmid: https://www.addgene.org/68581/

    • Study: https://pubmed.ncbi.nlm.nih.gov/26406247/

    • Pro Tip: Transform Efficiency. Biological processes are probabilistic. For us, the vast vast majority of bacteria carrying our payload will NOT gene transfer to a plant cell. So we want to do whatever we can to improve our odds, one of them being picking plasmids containing genes that have the highest rate of infection (aka infection with gene transfer). Here’s an example of that from the study

      

• Knowing Where to Add our Genetic Payload: “Left Border” & “Right Border” DNA sequences noting container for our DNA payload

  • Specific for agrobacterum, is the region of the plasmid that will be our gene payload transferred to the plant. Conveniently for us, this is easy to spot in annotations, as the right and left borders. What DNA we stitch into there will show up in our plant
  • show picture of borders

Picking a “reporter” gene payload: What to insert between left & right pMpGWB127 plasmid borders:

• Reporters are short hand for saying, some indicator that our genetic transformation worked. Often times they’re visual, in the form of fluorescent proteins.
  • It’s worth noting that a “reporter” can be one or multiple genes working in unison.
• Our Choice: RUBY

  • We’re going to use the RUBY reporter, which will just make the plant red. Very convenient! No equipment necessarily.

    s41438-020-00390-1.pdf

  • And additionally, why we’re picking it is because the plasmid used in this study is available for us on Addgene, via UBQ:RUBY (UBY in this shorthand is referring to the promoter, which we’ll talk about in a second)

    • Addgene: https://www.addgene.org/160909/
    • Study: https://pubmed.ncbi.nlm.nih.gov/33024566/
• DNA Fragments to Add:

  • reporter DNA sequence(s) for genes (which later can translate to proteins)

    • We will be using the reporter RUBY, which is actually a protein that appears due to interactions between 3 separate proteins, CYP76AD1, DODA, and Glucosyl transferase. So our “reporter” is going to be actually 3 separate but sequential genes.

    

  • promoter DNA: this is DNA sequence that corresponds to some organism/environmental trigger. This is what causes the plant cell to say ‘make the following protein’

    • We’ll use: Ubiquitin
      • it’s a common promoter across eukaryotic cells
      • it’s already proven to work in another plant via the study we have (however, the plant is arabidopsis, not marchantia)
      • It’s already incorporated in the plasmid

  • terminator: the DNA s

    • We’ll use: HSP
      • Lots of literature in arabidopsis plants, shown to increase efficiency
      • It’s already incorporated in the plasmid that demonstrated UBQ:RUBY to work

  • Pro Tip: These are unique to your organism. A promoter that triggers a fluorescent protein in bacteria will likely not work in a plant.