UNL iGEM 2018 Early Detection of the Emerald Ash Borer 1 What - - PowerPoint PPT Presentation

UNL iGEM 2018

Early Detection of the Emerald Ash Borer

1

What is synthetic biology?

• Interdisciplinary field
• Genetic Engineering
• Molecular Biology
• Computer Science
• Build and use biological parts called Biobricks
• Standardized gene blocks
• Biology equivalent of LEGO bricks

2

Biobrick Plasmid (n.d.). Retreived from http://agapakis.com/hssp/biobricks.html

What is synthetic biology?

• Use E. coli or Saccharomyces cerevisiae (baker’s yeast) as chassis
• Grown easily and inexpensively
• Rapid reproduction (40 minute life cycle)
• Engineered strain of E. coli that is non-pathogenic

3

• E. coli under a microscope

https://www.britannica.com/science/E-coli

What is iGEM?

• International Genetically Engineered Machines
• Synthetic biology competition
• Build genetically engineered biological systems with

interchangeable parts

• Solve real world challenges

4

What is iGEM?

340 teams from around the world work on their own projects

5

What is iGEM?

Project tracks:

• Diagnostics
• Energy
• Environment
• Food & Nutrition
• Foundational Advance

6

• Information Processing
• Manufacturing
• New Application
• Therapeutics

http://2018.igem.org/Competition/Tracks

7

UNL iGEM team

• UNL’s team’s third year participating in the event
• 2016 - Awarded Silver

○ 2017 - Awarded Gold ○ Environmental Track each year

20 2017 17

UNL iGEM Team

Choosing a project

• Area of concern or problem that Nebraska is facing
• Brainstorm how synthetic biology could solve the problem
• r improve on the current solutions

Emerald Ash Borer

8

9

• The Emerald Ash Borer (EAB) is an invasive species from Asia that first appeared in

the United States in Michigan in 2002

• Adult EAB lay eggs on the bark of ash trees (Fraxinus spp.)
• When the larva hatch, they bore into the trees
• Emerald ash borer (EAB) infests and kills all types of ash trees regardless of their

health, condition, size or age

Emerald Ash Borer

City of Longmont, Colorado. (n.d.). Retrieved from https://www.longmontcolorado.gov/departments/departments-e-m/forestry/emerald-ash-borer Foresters monitoring the emerald ash borer on the Oak Ridge Reservation https://phys.org/news/2014-10-foresters-emerald-ash-borer-oak.html (accessed Jun 19, 2018).

Infestation Timeline

10

Emerald Ash Borer Awareness (n.d.). Retreived from https://www.treecarescience.com/articles/news/emerald-ash-borer-awareness

EAB Movement

As of September 2018, the EAB has spread to 34 U.S. states and 4 Canadian provinces

11

http://www.emeraldashborer.info/about-eab.php

12

EAB Movement

Current State of the Emerald Ash Borer in Nebraska

• Confirmed in Omaha and

Greenwood in 2016

• Confirmed in Lincoln in

August 2018

University of Nebraska-Lincoln. “Nebraska Emerald Ash Borer.” Nebraska Emerald Ash Borer | Nebraska Forest Service, nfs.unl.edu/nebraska-eab. University of Nebraska-Lincoln. “Nebraska Emerald Ash Borer Quarantine Maps.” Nebraska Emerald Ash Borer | National Demolition Association, http://www.nda.nebraska.gov/plant/entomology/eab/

EAB Movement

• Natural EAB spread: 3 miles per year
• Distance between Nebraska and Michigan: >700 miles
• It arrived in Nebraska in only 15 years, so humans must have helped spread it
• Human spread is mainly from

moving infested firewood

13

https://vtinvasives.org/get-involved/slow-spread/dont-move-firewood

14

• Nebraska has 44 million ash

trees

• Estimated cost per 1 million

trees in Nebraska communities is $961 million

• Estimated cost of Lincoln’s

12,000 publicly owned street trees is $23,177,963 over 13 years

Pay Now, Save Later

Berg, E.; Bohall, B.; DuPlissis, J.; Erixson, J.; Herbst, G.; Marcum, D.; McCartney, F.; Pineo, H.; Sieber, L.; Smith, A.; Schluckebier, J.; Stansberry, J.; Young, L. Annual Report 2017; rep.; University of Nebraska-Lincoln.

• Nebraska Emerald Ash Borer Response Plan, May 15, 2015

https://nfs.unl.edu/NebraskaEABResponsePlan.pdf

Ohio Monetary Impact

15

Abell, K.; Poland, T.; Cosse, A.; Bauer, L. Biology and Control of Emerald Ash Borer 2014. Woda, M. What is That Purple Thing Hanging in The Tree? https://activerain.com/blogsview/1279581/what-is-that-purple-thing- hanging-in-the-tree- (accessed Jun 15, 2018)

• Early detection is important to monitor and limit EAB spread
• Visible signs of infestation only appear after EAB is firmly established
• Girdling a trap tree is an effective detection method,

but it requires stripping the tree’s bark and later cutting it down

• Traps are a nonintrusive way of detecting EAB
• Locate new infestations
• Risk assessment
• Population Levels

EAB Trap Importance

http://broomfield.granicus.com/MetaViewer.php?view_id=6&clip_id=1270& meta_id=36705

16

https://www.nrs.fs.fed.us/disturbance/invasive_species/eab/risk_detection_spread/trapping_attractants/

• Color
• Purple attracts females
• Green attracts males

EAB Trap Attractants

• Chemical lures
• Green leaf volatile cis-3-hexenol, currently used in traps (4)
• Manuka and Phoebe tree oils, which contain compounds

emitted by the bark of stressed ash trees commercially unavailable

EAB Trap Attractants

Research shows

• Traps with manuka oil and cis-3-hexenol catch more EAB than traps

with only cis-3-hexenol (2)

• Traps with phoebe oil catch nearly 2 times as many EAB than traps

with manuka oil (2)

Phoebe oil contains 7-epi-sesquithujene, which is considered to be the most attractive known compound to EAB, but manuka oil does not (2)

17

18

iGEM Project 2018

Project Goal: in vivo biosynthesis of 7-epi-sesquithujene

• 7-ES is a sesquiterpene, a type of compound known for being difficult to extract and

isolate from plants

• It is commercially unavailable since it is impractical to chemically synthesize (2)
• Research shows that E. coli can express a terpene synthase enzyme (TPS4-B73)

which has produced 7-ES among other products in vitro (3)

19

• Biosynthesis of 7-ES requires the precursor farnesyl pyrophosphate (FPP) (3)
• FPP can be produced in E. coli by engineering the mevalonate-dependent pathway

found in Saccharomyces cerevisiae and some enzymes from E. coli itself

• Using this pathway and the TPS4-B73 gene, E. coli can independently produce 7-ES (1)
• From there, the 7-ES could be purified to use in EAB traps

iGEM Project 2018

20

• E. coli Parts

Mevalonate-dependent Pathway

Anthony, J. R.; Anthony, L. C.; Nowroozi, F.; Kwon, G.; Newman, J. D.; Keasling, J. D. Metabolic Engineering2009, 11(1), 13–19.

21 FPP

Synthesis Pathway

TPS4-B73 Enzyme

Anthony, J. R.; Anthony, L. C.; Nowroozi, F.; Kwon, G.; Newman, J. D.; Keasling, J. D. Metabolic Engineering2009, 11(1), 13–19.

7-epi-sesquithujene

Experiments

• Sequence and Ligation Independent Cloning (SLIC) to clone the TPS4-B73 gene block
• Restriction Cloning to clone a codon-optimized TPS4-B73 gene block
• Developed a sesquiterpene production assay
• Overlay octane on cultures to collect sesquiterpenes through organic phase

separation

• Research shows the organic phase traps over 97% of the sesquiterpenes

produced (1)

• Analyze octane samples through gas chromatography-mass spectrometry to

identify and quantify products

22

Results

Codon-optimized TPS4-B73 GC Overlay

23

Total ion chromatogram

Selective ion search for the sesquiterpene parent ion 204 m/z

7-epi-sesquithujene sesquithujene beta-bisabolene

Results

7-epi-sesquithujene identification

24

Results

Beta-bisabolene identification

25

Results

Internal Standard Quantification

• No pure 7-epi-sesquithujene is available to use as a quantification standard
• Use a similar sesquiterpene, trans-beta-caryophellene, as an internal standard to compare

concentrations at different time points and estimate total concentration

• Able to confirm concentration is increasing over time until the 48 hr time point
• Rough estimate of 6 μg/mL 7-epi-sesquithujene

26

7-epi-sesquithujene Internal standard Trans-caryophellene Beta-bisabolene

Future Research

• Purify 7-epi-sesquithujene
• Optimize sesquiterpene production
• Study the byproducts of TPS4 synthase

27

Outreach

In addition to providing research experience, the iGEM program also provides

• utreach and leadership opportunities for its students

28

Exhibit at Morrill Hall

TEDxYouth Conference Activity

Outreach

Field trip with the Nebraska Forest Service

29

• The Emerald Ash Borer is a problem that Nebraska is currently facing
• The iGEM program allowed us to explore how synthetic biology may be

able to help deal with the EAB

• Successful in vivo synthesis of 7-epi-sesquithujene and future research

plans to continue studying sesquiterpenes

• Engaged in public outreach opportunities to share our iGEM project with

the community

30

Conclusion

• 1. Anthony, Jennifer R., et al. “Optimization of the Mevalonate-Based Isoprenoid Biosynthetic Pathway in Escherichia Coli for Production of

the Anti-Malarial Drug Precursor Amorpha-4,11-Diene.” Metabolic Engineering, vol. 11, no. 1, 2009, pp. 13–19., doi:10.1016/j.ymben.2008.07.007.

• 2. Crook, Damon J., and Victor C. Mastro. “Chemical Ecology of the Emerald Ash Borer Agrilus Planipennis.” Journal of Chemical Ecology,
• vol. 36, no. 1, 2010, pp. 101–112., doi:10.1007/s10886-009-9738-x.
• 3. Köllner, Tobias G., et al. “The Variability of Sesquiterpenes Emitted from Two Zea mays Cultivars Is Controlled by Allelic Variation of

Two Terpene Synthase Genes Encoding Stereoselective Multiple Product Enzymes.” The Plant Cell, vol. 16, no. 5, Sept. 2004, pp. 1115– 1131., doi:10.1105/tpc.019877.

• 4. Mccullough, Deborah G., and Therese M. Poland. “Building Double-Decker Traps for Early Detection of Emerald Ash Borer.” Journal of

Visualized Experiments, no. 128, Apr. 2017, doi:10.3791/55252.

32

References

33

Thank you!