Introduction: How to Grow “Hello World” With GFP Bacteria
Using bacteria as pigments to create "living-images" has been used by many artists and scientists. From the early petri-plate paintings of Alexander Fleming to the bacterium photographs of Edgar Lissel.
Taking inspiration from traditional and DIY biology combined with printmaking techniques, you can grow living design patterns!
In the following steps I will use a non-harmful E.coli bacteria that was genetically modified with a green fluorescent protein (GFP) extracted from the Aequorea victoria jellyfish.
Step 1: Gather Materials and Equipment
1. Petri-plate (100 mm diameter by 15 mm height)
2. Your favorite microorganism*
3. Your microorganism's favorite nutrient Agar
5. 70% rubbing alcohol isopropyl
7. Beakers / plastic containers x 3
8. Distilled Water
9. 10% Commercial Bleach
9. Assorted makeup sponge
10. 15ml tube
11. Tube rack
13. Waste bin
14. Acetate sheet (.005 clear film)
15. Kim Wipes / Paper towel
Step 2: Space and Sterilization
Use a clean STERILE hood or clean sterile bench area at your lab!
*Alternatively you can use a DIY hood made out of plastic container, or any non-absorbent surface area.
Clean your area with 70% rubbing alcohol and place your items.
You can also spray rubbing alcohol on your items before using them and on your gloves.
*For lab equipment or alternatively DIY equipment please advice with your scientific adviser or your local community lab.
Step 3: Lable Your Plate
Take one of your sterile petri-plates and while still closed flip it upside down. Use a sharpie pen and lable the BOTTOM of your petri-plate with your "Name", "Date", "Medium Type" and "Organism", in this order, along the circular edge of the plate. So your experiment will still be visible.
Step 4: Pour the Agar
Prepare your *nutrient Agar then gently open the lid of the petri-plate and pour the Agar into the plate to cover the bottom. The small petri-plate in this example needs about 15ml, but you will need more or less if you use different size plates.
Wait for the Agar to solidify and flip the plate upside down to prevent condensation droplets on the agar medium.
Let it rest while you sterile your stencil.
In this experiment I use LB Agar (Luria Broth) with the antibiotic Ampicillin (AMP).
*Choose the organism you will use in this experiment and follow the protocol to make its favorite nutrient Agar.
Step 5: Design Your Stencil
Measure your plate and create your design with a simple illustration app.
For a standard petri-plate, as used in this example, measure 3.3" diameter circle.
Use a CNC laser cutter and a clear acetate paper to create your stencil.
In this experiment I used a Grafix Dura-Lar Clear Acetate Alternative (.005 clear film).
Step 6: Sterile Your Stencil - the Triple Dip
Disinfect your stencil:
1. Place the stencil in a container /or a beaker with 10% commercial bleach (1/4 cup bleach + 2 1/4 cup water + few drops of dish detergent) and soak it for 10-15 minutes. Stir occasionally using a long forceps or spatula.
2. Transfer the stencil with tweezers to a container with distilled sterile water and soak for 2-3 minutes
to rinse off the bleach.
3. Finally, transfer the stencil with tweezers to a container with 70% rubbing alcohol for 1 min.
Wipe the alcohol with Kim Wipes or leave it to dry completely in a sterile area before applying it to the plate.
Step 7: Place Your Stencil
Flip your plate and carefully open the lid. Use the tweezers and gently place the sterile stencil on top of the solid agar. Make sure your stencil is placed on the right side to grow your design.
To avoid additional contamination, you can also place the plate with the stencil for 30 min under UV light before applying your microbial culture.
Step 8: Sterile the Sponge - Triple Dip
Use a wedge makeup sponge or cut a piece from your assorted sponges pack.
Disinfect your Sponge (Repeat Step 6):
1. Place the sponge in a container /or a beaker with 10% commercial bleach (1/4 cup bleach + 2 1/4 cup water + few drops of dish detergent) and soak it for 10-15 minutes. Stir occasionally using a long forceps or spatula.
2. Transfer the sponge with tweezers to a container with distilled sterile water and soak for 2-3 minutes to rinse off the bleach.
3. Finally, transfer the sponge with tweezers to a container with 70% rubbing alcohol for 1 min.
Leave the sponge to dry completely in a sterile area before applying it with bacteria onto the petri-plate.
Step 9: Apply Your Bacteria
Use the sterile sponge and soak it with your bacterial culture. Gently apply the sponge with the bacteria onto the exposed agar areas of your stencil.
You could also use a pipette to apply the bacteria onto the plate and then distribute the culture with the sterile sponge.
Let it sit and wait for a few minutes for the bacteria culture to dissolve. Flip the plate upside down to avoid condensation on the agar. Now you are all set for incubation.
Step 10: Incubation
Place the plate in a *37°C incubator for 1 or 2 days for the bacteria colonies to develop.
( Some microbes require 30°C incubator, check your protocols !)
After 1 or 2 days the bacteria colonies have developed and you will see
growth over the exposed areas of the agar where the microbes could easily feast on their favorite nutrients.
Step 11: Check Your Bacteria Design!
You can also check your plate under UV light if you used a fluorescent one.
Step 12: NYC Biome MAP
This method was used for the "NYC Biome MAP" as seen here with samples under UV light.
Step 13: Stay Tuned!
Next you will learn how to Print your designs !
Christine Marizzi, Sylvia Saborio, Ali Schachtschneider, Marta Molina Gomez