1. Discover the Microbes Within: Impacts of DNA-based technologies and PCR basics Seth Bordenstein Marine Biological Laboratory April 11, 2008

2. ARCHAEA BACTERIA EUKARYOTE C. Woese Microbes in Eukaryotic Evolution Everything we can see

3. Bacteria Fun Facts Most microbes do not cause disease! 1 gram of soil = 10 million bacteria More bacteria in your mouth than there are people in the world # beneficial bacteria in human intestine = # of cells in human body Oldest forms of life on earth are bacteria (3.8 billion years ago) Bacteria make up most of the biomass on earth, but only 1% have been cultured

4. Classical microbiology – phenotypic approach Culture Gram stain Shapes

5. Molecular microbiology – genotypic approach Identifies genes More accurate, objective, and reproducible results Identifies unculturable bacteria (99%) Eliminates special growth requirements requirements DNA sequence data are more easily shared and databased easily shared and databased Rapid diagnosis Resolves evolutionary relationships

6. Genes and Genomes: fun facts Bacteria have small genomes (1- 10 million base pairs vs. 3 billion base pairs in human) 1995: Entire genome of Haemophilus influenzae was sequenced Two years later: 12 genomes sequenced March 24, 2008: 747 genomes sequenced (613 are from bacteria) and 1753 ongoing bacterial genomes

7. Buchnera (0.64 Mb) Blochmannia (0.75 Mb) Wolbachia (1.27 Mb) Wigglesworthia (0.70 Mb)

8. Endosymbionts are the smallest bacterial genomes known Wernegreen 2002 Insect endosymbionts

9. Arthropods Nematodes Insects (20-75%!) How do we go from here…..? Crustaceans (35%) Chelicerates 2-6 million insect species are infected with Wolbachia!! Filarial nematodes (90%)

10. To studying the Wolbachia within? Credit: Mark Taylor

11. PCR Introduction Polymerase Chain Reaction (PCR) allows scientists to amplify minutes amount of a specific DNA sequence from a heterogenous DNA pool in a few hours Invented by Dr. Kary Millis in 1983 (Nobel Prize in Chemistry in 1993) One of the most widely used biotechnology techniques in biological research. PCR is the method of choice for symbiont detection.

12. Basics of PCR Template DNA - the starting DNA of interest. High temperature denatures template DNA into single strands and synthetic sequences of ssDNA (20-30 nucleotides) serve as primers Two different primers are used to bracket the target gene to be amplified DNA polymerase copies the complimentary strand starting at the primer. In one cycle, two identical strands are made.

13. To perform your PCR - Ready Beads Small quantity of DNA Primers Buffered solution containing DNA polymerase Four base pairs of DNA Cofactor MgCl 2 All in test tube Buffered solution containing DNA polymerase Four base pairs of DNA Cofactor MgCl 2 Small quantity of DNA Primers

14. Temperature drives the reaction

15. Target gene: 16S rDNA Small ribosomal subunit involved in mRNA translation process Small ribosomal subunit involved in mRNA translation process Ancient molecule, functionally constant, universally distributed Ancient molecule, functionally constant, universally distributed Helps identify unknown bacterium to genus or species levels Helps identify unknown bacterium to genus or species levels Present in bacterial symbionts; eukaryote has very divergent copy that is named 18S rRNA Present in bacterial symbionts; eukaryote has very divergent copy that is named 18S rRNA

16. PCR Animation http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf

17. Two Key Innovations for Success of PCR Heat-stable DNA polymerase isolated from bacterium Thermus aquaticus which inhabits hot springs Polymerase remains active despite being heated many times DNA thermal cyclers – a computer that controls repetitive temperature changes required for PCR 70C hot springs in Yellowstone National Park Example of a thermal cycler from MJ Research

18. PCR and Pop Culture “Jurassic Park” and “CSI” “Jurassic Park” and “CSI” Some fun PCR facts to share with your students: …PCR has been used to amplify DNA from… a preserved quagga (a zebra relative that became extinct 100 years ago) crime scenes (e.g., O.J.) eight-celled human preembryos, to detect cystic fibrosis the brain of a 7000 year old American mummy patients for disease diagnosis

19. Our goal: Determine which of your insects harbor Wolbachia? DNA extraction: PCR: Gel electrophoresis:

20. Bellingham HS, MA Falmouth HS, MABronx HS, NY

21. One day till we discover the microbes within!

22. 2005 Workshop Participant 2007-2008 Student The Wolbachia Project: A “Bridge” to New Science in the Classroom:

23. Senior Student North Attleboro High School, MA “A Safer Alternative to Traditional Insecticides: Exploring the Applications of Wolbachia”  submitted to the JSHS Symposium at Boston University  selected to create a poster of her research  won an award for being the most creative project at the fair  won first place at the regional science fair  continues on to the state science fair in May

24. Progression of the Wolbachia Project Professional Development Professional Development New Science Implementation Summer Envisionships Summer Envisionships