Team:Bio Without Borders/Public Engagement


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JUDGING FORM ⇗

Public Engagement

Our team has worked with a high school teacher from Florida. We discussed the process of making a lesson plan for students. This lesson plan will involve the students doing lab works of how to do certain lab protocols such as transformation or restriction digest. The lesson will involve them analyzing how the they could integrate these works in the lab to stopping this citrus disease. Children often have a big impact on how adults think. By motivating young people to seek out challenges and face problems it will also affect their parents.

HUANGLONGBING (HLB) UNIT

PART I: LABORATORY PRACTICE

STUDENT LAB INSTRUCTIONS

INTRODUCTION


An infectious citrus-greening disease devastating the orange industries of Florida, Huanglongbing (HLB) has become an urgent dilemma. This lab activity introduces students to the detection, identification, and quantification of plant pathogens like HLB in the stomach of the Asian Citrus Psyllids that transmit them. By testing collected samples of DNA from the psyllids, students can check to see if the gene sequences match that of the HLB-causing bacteria.

PREPARATION CHECKLIST

  • Gloves (latex)
  • Parafilm
  • Sample of Asian Citrus Psyllid
  • Extraction buffers  
  • Microcentrifuge with variable speed capable of 14,000 x g
  • Microcentrifuge tubes, 2 mL
  • 50x TAE, 100 mL
  • Weigh boats
  • Molecular biology grade agarose, 5 g
  • Ethanol
  • Graduated cylinders
  • Microwave oven
  • Horizontal electrophoresis chamber
  • Erlenmeyer flask
  • Distilled water
  • Water bath
  • Thermal cycler
  • Spin columns
  • Resuspension solution, 5 mL
  • Lysis solution, 5 mL
  • Neutralization solution, 5 mL
  • Matrix, 5 mL
  • Wash buffer, 10 mL
  • Master mix for PCR, 1.2 mL
  • PCR tubes, 0.2 mL
  • PCR tube racks
  • UV transilluminator
  • Pipette tips (2-20, 20-200, 100-1,000 microliters)
  • Adjustable micropipettes (2-20, 20-200, 100-1,000 microliters)

LAB PROTOCOL

  1. Sample preparation
  2. (Materials: Asian Citrus Psyllid sample)
  1. Acquire Asian Citrus Psyllid sample.
  2. Prepare a small piece of psyllid tissue.
  1. DNA extraction(Mini Prep)
  2. (Materials: Centrifuge, Prepared psyllid sample)
  1. Pellet 5 mL by centrifugation for 30 seconds at 4000 rpm. Discard supernatant into a liquid waste tube once centrifugation is complete.
  2. Resuspend pellet in 200 μL Plasmid Resuspension Buffer (B1). Pipette up and down to resuspend the cells.
  3. Add 200 μL Plasmid Lysis Buffer (B2), gently invert the tube 5-6 times and incubate at room temperature for one minute. A color change to dark pink should be observed.
  4. Add 400 μL of Plasmid Neutralization Buffer (B3), and gently invert tube until neutralized, and incubate at room temperature for two
  5. Centrifuge lysate for minutes
  6. Transfer supernatant to the spin column and centrifuge for one minute. Discard flow through.
  7. Re-insert column in the collection tube and add 200 μL of Plasmid Wash Buffer 1. Centrifuge for one.
  8. Add 400 μL of Plasmid Wash Buffer 2 and centrifuge for one minute.
  9. Transfer column to a clean 1.5 ml microfuge tube. Ensure that the tip of the column does not touch the flow
  10. Add 30 μL of DNA Elution Buffer to the center of the column. Let it sit for one minute and spin for one minute to elute the buffer.
  1. PCR amplification and electrophoresis
  2. (Materials: Master mix, Agarose gel, PCR materials)
  1. Add master mix to the psyllid DNA samples and control DNA.
  2. Amplify the targeted sequences by PCR.
  3. Prepare gel electrophoresis for 20 minutes in order to analyze the DNA.
  1. PCR results analysis
  2. (Materials: UV transilluminator, UView 6x loading dye)
  1. Observe the results on a UV transilluminator.
  2. For instantaneous results, use UView 6x loading dye.
  1. Sequencing and bioinformatics for species identification
  1. Send the PCR products to a sequencing facility to analyze the sequences using DNA bioinformatics platform.
  1. Bioinformatics analysis
  1. Discuss implications and results with students.

Source: Bio-Rad Explorer, p. 67

REFLECTION

  • Defend whether you believe citrus tree owners are justified in keeping them in their backyards, despite the prevalence of HLB.
  • How is biological information stored in living things and how we analyze and classify that information?
  • Why is knowing certain sequences of DNA essential to making primers and other enzymes?

RESULTS & DISCUSSION

The investigation should provide students with an idea of the process of identifying bacterial DNA sequences and its role in diagnosing Asian Citrus Psyllid for the HLB bacteria. Particularly considering the impact of the spread of HLB, it becomes invaluable for students to get hands-on experience with and become aware of the nuances of DNA sequencing in identifying HLB before it’s too late to implement procedures to save the citrus plant.

TAKE IT A STEP FURTHER

Now that you’ve really learnt more about HLB, take a stab at applying your knowledge to the real world.

  • Video: Film a short five-ten minute film depicting a public service announcement or documentary about HLB. Warn your community about its effects and urgency!
  • Letters: Write a short letter to your local officials (e.g. Environmental Club advisor, principal, mayor, state representatives, corporate organizations) explaining this predicament. Try to come up with possible policies that can address it.
  • Tip: Acknowledge that some families might be reluctant to part with their citrus trees. Also remember that no amount of change is too little. We’re working at it bit by bit!
  • Poster: Make a visual representation of your results and findings. Display it in your school or community center.
  • Story Book: Create a story book with a little plot incorporating HLB facts and have it published or circulated.  
  • Website: Establish your own website, communicating HLB and how site-viewers can help!  
  • Tip: Also make social media to connect with your domain. This will only increase the number of people you reach.
  • Come up with your own idea! There’s plenty more ways out there. Run it by your teacher and obtain approval beforehand!

INFORMATION FOR INSTRUCTOR

Additional Background for Teachers

Huanglongbing (HLB) is an infection of the bacterium Candidatus liberibacter asiaticus (CLas). It compromises the immune system of host citrus plants. As the asian citrus psyllid, Diaphorina citri, feeds on the plant’s sap, it introduces the disease. CLas migrated from the insect gut into the phloem through the psyllid’s saliva. Unfortunately, treating them with pesticides and antibiotics have disastrous long-term effects, such as ecological damage and development of antibiotic resistant soil bacteria.

We, the 2019 Bio Without Borders team, decided to create this unit plan to introduce students to citrus pathology and the concerns regarding the urgent HLB disease. Our team focuses on modifying the systems of the 2017 TecCEM team, silencing crucial psyllid developmental genes via siRNA, targeting arginine kinase and superoxide dismutase, and exploring the use of whole bacteria instead of purified siNRA to treat plants. We envision that we will be able to realize our objective by designing a trap containing RNase minus E.Coli/siRNA-producing cassettes.

Raising awareness begins in the classroom. We know that our first and most important step would be bringing hands-on activities to educational institutions. This will ensure that, if nothing else, the next generation will at least know about HLB and its terrifying consequences. And this leaves promise that we just might be able to change the world, one step at a time.

Concepts and Methods

  • To diagnose psyllids for HLB disease
  • To detect if DNA of bacteria is in stomach

The foundation of this lab exercise is largely based upon the following standards.

Our reflection questions are designed according to the Rigormeter standard, Level 6, meant to challenge students to fully master the content material. We not only ask them to comprehend, apply, and analyze the background, we also ask them to be able to synthesize and evaluate—in order to fully encourage the development of their thinking skills.

 

Source: Roberta Lenger Kang (2010) https://teachers.ash.org.au/researchskills/dalton.htm#knowledge

Lab Safety 

We always wear gloves and long sleeved clothes in the lab to prevent any chemicals coming in contact with our skin. Any material used in the lab must be placed in their original spot. Everything in the lab ideally should be labeled. The sample of Asian Citrus Psyllid should be controlled. Make sure students are not handling the ethidium bromide for the gel electrophoresis.

Problem-Based Learning  

At the conclusion of this lab, we assign a long-term project. This is motivated by a popular learning method known as problem-based learning. As a result of the problem of HLB (which they’re introduced to by participating in this assignment), we encourage them to tackle the situation by applying their skills. Students may wish to work either independently or in groups, either way familiarizing themselves the intricacies of real-world problem-solving skills—essential to students’ future careers and lives. This approach allows teachers to discover an answer for what exactly they want their students to take away from the project.

Source: Nilson, L. B. (2010). Teaching at its best: A research-based resource for college instructors (2nd ed.).  San Francisco, CA: Jossey-Bass.