Ending the Reproduction of Kudzu
By: Juliana Yagyu & Jordan Holdaway
Background
Kudzu is an invasive plant species that
may lead to a decrease in soil productivity
because of its high amounts of nitrogen. It
can also be found in unwanted places that
are bothersome to farmers and others that
work outdoors. In the United States it
costs around $500 million annually in lost
cropland and control costs to manage
kudzu. However, by using a CRISPR kit to
learn more about gene editing and
researching Kudzu this year we can use
our newly obtained knowledge to stop the
growth of Kudzu with CRISPR the following
year.
Research Question? / Hypothesis
Research Question(s): Will
Kudzu stop reproducing if its genome
is altered?
Hypothesis(es): If we research more
about Kudzu as well as the genes involved
in reproduction and use CRISPR to learn
how to edit genes then we can
stop the reproduction of Kudzu by
altering its genome.
Conclusions
The hypothesis was proven or disproven
because of a misstep made in our project.
We dropped a few plates on the ground
during testing and we scrapped the plate
too roughly when we were attempting the
last steps in our procedure.
Harrison High School
Materials :
• LB Agar
• LB Strep/Kan/Arabinose Agar
• Glass bottle for pouring plates
• Inoculation Loops/Plate Spreader
• 10-100uL variable volume
adjustable pipette
• (1 uL increments)
• Box of 96 Pipette Tips
• 14 Petri Plates
• Microcentrifuge tube rack
• Nitrile Gloves
• Microcentrifuge tubes
• 50mL Tube for Measuring
• Bacterial transformation buffer
25mM CalCl2, 10% PEG 8000
• LB Media for transformation
recovery
• Cas9 Plasmid
• gRNA plasmid
• Template DNA
• Bacterial Transformation Kit
Pictures of the Procedure & Results
This is when we made the
bacterial transformation
mix.
Materials and Procedure
Procedure:
1. Purchase a Bacterial Gene Engineering CRISPR
Cas9 Kit
2. Take 1 hour to make plates (it may take more
time if inexperienced in making plates)
3. Streak out bacteria onto an LB Agar plate for
about 1 minute
4. For 12-18 hours let the bacteria grow (it is best to
let it sit overnight)
5. The next day or when the bacteria have had time
to grow, mix together sample, plasmids, and
transformation mix
6. Refrigerate sample solution for 30 minutes (Do
NOT freeze!)
7. For 30 seconds, ‘heat shock’ the sample warm at
42 degrees celsius or 108 degrees Fahrenheit
water.
8. Add LB media to your cell solution for about 1
minute then incubate for at least 1-2 hours at 30
degrees celsius or incubate for at least 4 hours at
room temp. for the best results
9. Plate 200uL of the bacteria solution and let it dry
for 10 minutes
10. Incubate for about 24 hours at 30 degrees Celsius
or 86 degrees Fahrenheit for 16-24 hours or at
room temperature for 24-48 hours
A picture of the different This is our final product after plating
bacterial transformation our bacterial transformation mix onto
tubes. the LB Strep plate.
Next Steps
Our next steps are to use our knowledge
on CRISPR to modify the genome of kudzu
and make it stop reproducing. We now
know more about kudzu and how to use
CRISPR to prepare us for next year.
References
Bortesi, L., & Fischer, R. (2015). The CRISPR/Cas9 system for plant
genome editing and beyond. Biotechnology Advances, 33(1), 41-
52. Retrieved from https://www.sciencedirect.com/science/article/pii/
S0734975014001931
Crossley, M. (2018). What is CRISPR gene editing, and how does it
work? (Cover story). Chemistry in Australia, 18–19. Retrieved from
http://search.ebscohost.com/login.aspx?direct=true&db=a9h&AN=13
0038067&site=ehost-live
Jinek, M., Liang, P., Vyas, V. K., Barrasa, M. I., Fink, G. R., Maddalo, D.,
Yin, H, Oye, K. A., Gantz, V. M. & Bier, Ishino, Y., Shinagawa,
H., Makino, K., Amemura, M. & Nakata, A., Mojica, F. J., Ferrer,
C., Juez, G. & Rodríguez-Valera, F., Barrangou, R., Cong, L., Mali, P.
(2015). CRISPR, the disruptor. Nature. Retrieved from
https://www.nature.com/news/crispr-the-disruptor-1.17673
Liu, D., Hu, R., Palla, K., Tuskan, G., Yang, X. (2016). Advances and
perspectives on the use of CRISPR/Cas9 systems in plant genomics
research. Retrieved
from https://www.sciencedirect.com/science/article/pii/S1369526616
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Park, A. (2016). The CRISPR Pioneers. TIME Magazine, 188(25–26),
116–122. Retrieved from
http://search.ebscohost.com/login.aspx?direct=true&db=a9h&AN=12
0052882&site=ehost-live
Richard, G., G., P., Fossdal, G., C., P., W., & D., T. (2015, October 5).
Extreme low temperature tolerance in woody plants. Retrieved
from https://www.frontiersin.org/articles/10.3389/fpls.2015.00884/ful
l
Vyas, K. V., Oye, A. K., & Barrangou, R. (2015, June 4). CRISPR, the
disruptor. Retrieved from https://www.nature.com/news/crispr-the-
disruptor-1.17673
Zalatan, G. J., Polstein, R. L., & Hilton, B. I. (2016, March 10). CRISPR:
gene editing is just the beginning. Retrieved August 21, 2019,
from https://www.nature.com/news/crispr-gene-editing-is-just-the-
beginning-1.19510
Acknowledgements
We would like to thank many people for
helping us complete our science fair this
year, we couldn't have done it without
them. Specifically we would like to thank
Mrs. Curran, Ms. Allard, Mr. Ward, Mr.
Holder, Mrs. Tatum, and our parents.