Name: Marianne I.

Gugulan Subject: General Biology 2

Glowing Plant
Materials and equipment:

• Vial of Glowing Plant DNA

• Pre-poured LB plates
• Arabidopsis Seeds
• Agro-bacterium
• Silwet
• Nutrient Solution
• Petri Dish
• Disposable Pipettes
Steps and Procedure:
1) Phase 1: Grow the plants
a) Arabidopsis is known as the fruit fly of plant biology due to its fast growth.
2) Phase 2: Transform the bacteria
a) Obtain the Glowing Plant DNA (GFP - fluorescent).
b) Insert the GFP DNA into the Agro-bacterium, which acts as a transport vehicle.
c) Use a process called cold snap transformation, which involves moving the bacteria from a very
warm environment to a very cold environment for 5 minutes, then back to a very warm
environment.
d) The changing temperatures cause pores to open up in the bacteria, allowing the new DNA to
enter.

Preparation Step: Grow Bacteria

i) First, add blocks of dry ice to a beaker filled with 500ml of isopropyl alcohol to create a dry
ice bath. The goal is to get the bath to -78 degrees Celsius – the sublimation point of CO2.
ii) Using a pipette, scrape off about a pea-sized amount of the bacteria to a small tube (called a
microcentrifuge tube) filled with 75 mM (millimolar) of calcium chloride to prepare the
bacteria to be frozen and thawed.
iii) Transfer a small amount of the cells (approximately 100 microliters) to a new tube, add the
DNA (we were using GFP, not the glowing plant DNA) to the bacteria, and include a negative
for experimental purposes.
iv) Put the tubes into the incubator, which should be set at 37 degrees Celsius. Keep the bacteria
mixed with DNA in the incubator for 5 minutes to warm the bacteria up.
v) 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 you are using isopropyl rubbing alcohol which is used to kill bacteria.
vi) Quickly move the tubes back to the 37 degree Celsius incubator bath. Incubate for an
additional 5 minutes.
vii) hake 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.
viii) 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.
Name: Marianne I. Gugulan Subject: General Biology 2

ix) Take a couple of the single circles of bacteria and spread out on 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.
3) Phase 3: Transform the plant
a) Trim the Plant. Carefully cut off all the blooming seeds or flowers you find on your Arabidopsis
plant. Kyle told me to cut off anything that looked white-ish. Be sure to leave buds that haven’t
bloomed.
b) 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.)
c) Dip. Dip the ends of the plant for five minutes.
d) Wrap it up! Gently wrap the plant in Saran wrap to increase the humidity.
4) Phase 4: Grow seeds
a) When the new seeds blossom, check them for a faint glow. Harvest those seeds, grow the plants,
and continue the process again.

Benefits of Glowing Plants:

1.) Save energy and Sustainability
Imagine if we could replace street lamps with glowing trees that require minimal maintenance and no
energy input. Not only would it result in significant energy savings, but these living organisms could
provide lighting for centuries to come, given their lifespan of more than 100 years. It's a sustainable
solution that benefits both the environment and future generations.

2.) Light source is attainable

Glowing plants have the potential to serve as a natural alternative to artificial lighting, which could reduce
our overall energy consumption. In addition to being more sustainable, this technology could also provide
nighttime illumination to those living without access to electricity.
Name: Marianne I. Gugulan Subject: General Biology 2

Glowing Tree
Steps and Procedure:
1) We use software called Genome Compiler to search for gene sequences and modify them in a
graphical interface.
2) Using this software, we look up Vibrio fischeri genes and optimize their sequences to work in plants
instead of bacteria.
3) We synthesize the DNA and print it using the software. The file is then emailed to a company that
makes the DNA for us.
4) Once we receive the DNA from the company, we insert it into a type of bacteria called agrobacterium,
which can do genetic engineering on its own. This bacterium inserts the DNA into the female gametes
of the plant.
5) We grow the seeds that come from those flowers, and the DNA we designed on the computer will be
present in the plant.
6) We also use a gene gun, which is a device that fires the DNA at high velocity into the cells of the
plant. Some of the cells will absorb the DNA and begin to express it.

Benefits of Glowing Trees:

1) Introducing bioluminescent species into cities can improve biodiversity and create a safe environment
for pollinators such as bees, butterflies, and birds. This can lead to the establishment of green spaces
in cities that may otherwise be inhospitable to wildlife, resulting in more sustainable ecosystems
overall.
2) Increasing the amount of carbon dioxide absorption from the atmosphere while reducing fossil-fuel
dependence to power public lights can be achieved by planting bioluminescent trees. Moreover,
bioluminescent lights have a wider range of applications beyond public areas. They can be used
inside pots and containers in homes, offices, and apartments to reduce the need for electricity.
Additionally, the use of organic light sources can help in mitigating the heat produced by artificial
lights.
3) Bioluminescent trees can help improve security in urban areas, particularly for pedestrians walking at
night. Illuminated areas are known to have lower crime rates, which makes the use of bioluminescent
trees a great solution for enhancing security.

From supporting local wildlife to enhancing urban safety and creating more sustainable ecosystems.