One of the most ambitious (and controversial) projects to explode on Kickstarter recently has been the Glowing Plant project. Nearly 6,000 backers have joined the campaign to use synthetic biology to create plants that will, hopefully, replace your desk lamp. So far, that level of brightness is just a dream, but the prospects for creating a plant that actually glows (however faintly) is very real. And Antony Evans, Kyle Taylor, and Omri Amirav-Drory want to take you along for the journey.
But really what they’re doing is creating a new kind of plant in a hackerspace.
The project is not without its detractors and skeptics. A small number of environmental groups are raising concerns about the project, demanding that the group abandon the project. The New York Times had an excellent write-up that weighed all the different perspectives.
Me? I’m curious. I’ve listened to arguments and explanations from both sides. And after doing my research, I tend to agree and support the Glowing Plant’s stated mission:
“Our primary goal with the project is to inspire and educate the general public regarding the potential benefits of this technology. It is our opinion that this technology should not be the exclusive preserve of large corporations but that the benefits it brings should be available to everyone.”
But if I’ve learned anything over the past few years, it’s that to really understand something, you have to make it yourself. So that’s what I did (or tried to do). Last week, I made the trip to BioCurious (the Kickstarter-funded community bio lab in Sunnyvale), to go through many of the steps with the project creators, and get a sense for how accessible this was from the perspective of a complete beginner.
What You Need
At the $250 reward level, Kickstarter backers can pledge for the DIY Maker Kit, most everything you need to recreate the project at your school or community biolab like BioCurious or Genspace. Includes: - Vial of Glowing Plant DNA - Pre-poured LB plates - Arabodopsis Seeds - Agro-bacterium - Silwet - Nutrient Solution - Petri Dish - Disposable Pipettes
What you’ll need that the kit doesn’t include: Dry Ice (available from your local grocery store) Isopropyl Alcohol Domestic Bleach (used to dispose of biological waste)
Note: Like many bacteria, agrobacterium is NOT a human pathogen. It is, however, still important when working with the bacteria to use disposable gloves which acts as an added layer of safety. Additionally, anything that has come in contact with the bacteria should be soaked in bleach for 30 minutes to kill any bacteria. As DIYBiologists, it is important to be safe and dispose of waste properly.
To be clear, I had no idea what most of this stuff was. I had never used a pipette before. I didn’t know what LB was (lysogeny broth – basically bacteria food). I don’t have a background in biotechnology, so the whole experience was new. In addition to the steps required for the project, I was learning basic safety and lab protocols. In fact, that was my biggest takeaway from the experience. Everyone at BioCurious is very conscious of safety and cleanliness. If this is your first time doing a DIYBio project, finding a space like BioCurious to get you up to speed on lab protocols is the prerequisite step.
Phase 1: Grow the Plant! Timeline: 4-8 Weeks of Grow Time Arabodopsis is the fruit fly of plant biology, or so I was told. It grows fast, making it easier and more conducive to the type of experiment we were running. Lucky for me, Antony and Kyle had pre-grown the Arabodopsis plants for the day’s experiment. Thank goodness, too, because I’m not known for my green thumb.
Phase 2: Transform the Bacteria Time Required: 3 hours This is the phase where you insert the Glowing Plant DNA (we were using GFP – fluorescent, not bioluminescent, as the new Glowing Plant DNA hasn’t been created yet) into the Agro-bacterium. The agro-bacteria is like the transport vehicle. This type of genetic manipulation isn’t new. Plant molecular biologists discovered this bacteria in the late 70′s and we were using this same natural process with new synthetic DNA.
To transform the bacteria, we used a process called a cold snap transformation, meaning we move the bacteria from very warm, to very cold (for 5 minutes), then back to very warm. The changing temperatures cause pores to open up in the bacteria and the new DNA to get in.
Preparation Step: Grow Bacteria Before you can transform the bacteria, you’ll need to grow the bacteria. The bacteria will come freeze dried (lyophilized for those who like jargon). Add a bit of sterile water to rehydrate then spread the bacteria on the LB plate (solid bacteria food) and keep them in the dark (covering with aluminum foil is good way to go). Leave the bacteria sit at room temperature and grow for 5-7 days. Most likely, you would need a few days to recover after being freeze dried too.
Step 1: Prepare the Dry Ice Bath Add blocks of dry ice to a beaker filled with 500ml of isopropyl alcohol. The goal is to get the bath to -78 degrees Celsius – the sublimation point of CO2.
Step 2: Prepare the Bacteria to be frozen and thawed. Using the pipette, scrape off about a pea size amount of the bacteria to a small tube (called a microcentrifuge tube) filled with 75 mM (millamolar) of calcium chloride.
Step 3: Transfer a small amount of the cells (approximately 100 microliters) to a new tube. And add the DNA (again, we were using GFP not the glowing plant DNA) to the bacteria.
We also included a negative for experimental purposes.
Step 4: Put the tubes into the incubator. You should have an incubator bath of 37 degrees Celsius. Keep bacteria mixed with DNA in the incubator for 5 minutes to warm the bacteria up.
Step 5: Move to the dry ice bath Quickly move the tubes to the dry ice bath. The quick change in temperature is what cracks open the cells. Incubate for 5 minutes, making sure the solution doesn’t get into the test tubes because remember you are using isopropyl rubbing) alcohol which is used to kill bacteria.
Step 6: Back into the incubator Again, quickly move the tubes back to the 37 degree Celsius incubator bath. Incubate for an additional 5 minutes.
Step 7: Shake! You’ll need to shake the tubes for between 4 and 5 hours. If you don’t have one of the crazy shaking machines that they have at BioCurious, you can set the tubes in a container on top of your washer or dryer for a similar amount of time. (I’m actually making that up, but it also might work.)
Step 8: Plate! After shaking, spread the bacteria out on the other pre-poured LB plate. Cover and incubate at room temperature for about 7 days. Small circles of bacteria should grow up which should be the bacteria that have the DNA in them.
Step 9: Prepare for transformation Take a couple of the single circles of bacteria and spread out on a another pre-poured LB plate. Cover and grow for about 5-7 days until a nice lawn of bacteria grows up. This lawn is what will be used for the plant transformation.
Phase 3: Transform the Plant Time Required: 60 minutes
Step 1: Trim the Plant Carefully cut off all the blooming seeds or flowers you find on your Arabodopsis plant. Kyle told me to cut off anything that looked white-ish. Be sure to leave buds that haven’t bloomed.
Step 2: Prepare the Glowing Plant Bacteria Add your newly created bacteria to a container filled with 200ml of water and 10 g of sugar (for the bacteria to eat.)
Step 3: Dip Dip the ends of the plant for five minutes.
Step 4: Wrap it up! Gently(!) wrap the plant in Saran wrap to increase the humidity.
Phase 4: Grow the seeds! Time required: 1 month When the new seeds blossom, check them for a faint glow. Harvest those seeds, grow the plants, and continue the process again!
Note: Release of plants transformed with agrobacterium is strictly regulated. This is important given that agrobacteria is a plant pathogen (even if it is disabled). You are now a DIYBiologist and it is important we are all good stewards and adhere to safety regulations of the technology to make sure it is available to others.
If you want to learn more about the Glowing Plant project, you can get more information on their Kickstarter page or come and meet them at Maker Faire (Meet the Makers Stage on Saturday, 1:30pm) this weekend!