This is a step-by-step hand holding, descriptive, illustrated picture guide for gene editing marchantia spores using aggrobactereum to become a purple color. This is a continuation of the Botanical Pharmacy where we will grow insulin in the marchantia plant.
Why? When trying to insert the insulin gene, it can take a lot of work to verify it’s there. So we’re going to add it to a larger plasmid that also expresses a red/purple color using the RUBY gene. This guide is for doing just the RUBY plasmid backbone insertion, proving we can transform the plant. In the guide following, we’ll add our insulin.
Example of our final plate. The purple plants contain the RUBY backbone plasmid!
We don’t want “chimeric” plants, only those will full transformation
Start → End: 7 Days (a few periods of overnight incubation), then 14 Days
To transform marchantia spores, we have to think in terms of two time lines: 1) spores and 2) aggrobactereum. Spores will be incubated for 5 days, and in that time we’ll streak agrobactereum containing our UBY:RUBY (2 days) and incubate it (2 days) so it can transform our spores on day 5. It’s good to be aware of those two separate timelines that meet up on day 5.
| Stage | Time/Days | Schedule |
|---|---|---|
| Stage 1: Spore Sterilization & Culture | Saturday (Week 1) | |
| Wait… | 3 Days | |
| Stage 2: Agrobacterium Culture | Tuesday (Week 1) | |
| Wait… | 2 Days | |
| Stage 3: Inducing Agrobacterium | Thursday (Week 1) | |
| Wait (Same Day) | 6 Hours | |
| Stage 4: Co-Culture Spores + Agrobacterium | Thursday (Week 1) | |
| Wait… | 2 Days | |
| Stage 5: Strain & Plate Spores on Selection | Saturday (Start of Week 2) | |
| Wait… | 1-2 Weeks | |
| Stage 6: Re-plate transformed Marchantia | Saturday (Week 4) |
Before we begin, let’s think about where we want to end up. If we’re trying to create a stock of plasmids containing GFP and ampicillin genes, how much stock do we want/need?
Materials