UNL iGEM 2018 Early Detection of the Emerald Ash Borer 1
What is synthetic biology? • Interdisciplinary field Genetic Engineering o Molecular Biology o Computer Science o • Build and use biological parts called Biobricks Standardized gene blocks o Biology equivalent of LEGO bricks o 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 E. coli under a microscope https://www.britannica.com/science/E-coli 3
What is iGEM? • International Genetically Engineered Machines • Synthetic biology competition o Build genetically engineered biological systems with interchangeable parts o 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: • Information Processing • Diagnostics • Manufacturing • Energy • New Application • Environment • Therapeutics • Food & Nutrition • Foundational Advance http://2018.igem.org/Competition/Tracks 6
UNL iGEM team • UNL’s team’s third year participating in the event o 2016 - Awarded Silver ○ 2017 - Awarded Gold 20 2017 17 ○ Environmental Track each year 7
UNL iGEM Team Choosing a project • Area of concern or problem that Nebraska is facing • Brainstorm how synthetic biology could solve the problem or improve on the current solutions Emerald Ash Borer 8
Emerald Ash Borer ● 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 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). City of Longmont, Colorado. (n.d.). Retrieved from https://www.longmontcolorado.gov/departments/departments-e-m/forestry/emerald-ash-borer 9
Infestation Timeline Emerald Ash Borer Awareness (n.d.). Retreived from https://www.treecarescience.com/articles/news/emerald-ash-borer-awareness 10
EAB Movement As of September 2018, the EAB has spread to 34 U.S. states and 4 Canadian provinces http://www.emeraldashborer.info/about-eab.php 11
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/ 12
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 https://vtinvasives.org/get-involved/slow-spread/dont-move-firewood 13
Pay Now, Save Later Ohio Monetary Impact ● 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 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 14
EAB Trap Importance ● 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 o Locate new infestations o Risk assessment o Population Levels 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- http://broomfield.granicus.com/MetaViewer.php?view_id=6&clip_id=1270& hanging-in-the-tree- (accessed Jun 15, 2018) meta_id=36705 15
EAB Trap Attractants • Color o Purple attracts females o Green attracts males • Chemical lures o Green leaf volatile cis-3-hexenol, currently used in traps (4) o Manuka and Phoebe tree oils, which contain compounds emitted by the bark of stressed ash trees commercially unavailable https://www.nrs.fs.fed.us/disturbance/invasive_species/eab/risk_detection_spread/trapping_attractants/ 16
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
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) 18
iGEM Project 2018 ● 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 19
E. coli Parts Mevalonate-dependent Pathway Anthony, J. R.; Anthony, L. C.; Nowroozi, F.; Kwon, G.; Newman, J. D.; Keasling, J. D. Metabolic Engineering 2009, 11 (1), 13 – 19. 20
Synthesis Pathway TPS4-B73 FPP 7-epi-sesquithujene Enzyme Anthony, J. R.; Anthony, L. C.; Nowroozi, F.; Kwon, G.; Newman, J. D.; Keasling, J. D. Metabolic Engineering 2009, 11 (1), 13 – 19. 21
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 Total ion beta-bisabolene 7-epi-sesquithujene sesquithujene chromatogram Selective ion search for the sesquiterpene parent ion 204 m/z 23
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 μ g/mL 7-epi-sesquithujene Internal standard 7-epi-sesquithujene Trans-caryophellene Beta-bisabolene 26
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 outreach and leadership opportunities for its students Exhibit at Morrill Hall TEDxYouth Conference Activity 28
Outreach Field trip with the Nebraska Forest Service 29
Conclusion • 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
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