Discover the Microbes Within:Discover the Microbes Within:Impacts of DNA-based Impacts of DNA-based

technologies and PCR basicstechnologies and PCR basics

Seth BordensteinSeth Bordenstein

Marine Biological LaboratoryMarine Biological Laboratory

April 11, 2008April 11, 2008

ARCHAEAARCHAEA

BACTERIABACTERIA

EUKARYOTEEUKARYOTE

C. WoeseC. Woese

Microbes in Eukaryotic EvolutionMicrobes in Eukaryotic Evolution

Everything we can seeEverything we can see

Bacteria Fun FactsBacteria Fun Facts

Most microbes do not cause disease!Most microbes do not cause disease!1 gram of soil = 10 million bacteria1 gram of soil = 10 million bacteriaMore bacteria in your mouth than there are More bacteria in your mouth than there are people in the worldpeople in the world# beneficial bacteria in human intestine = # of # beneficial bacteria in human intestine = # of cells in human bodycells in human bodyOldest forms of life on earth are bacteria (3.8 Oldest forms of life on earth are bacteria (3.8 billion years ago)billion years ago)Bacteria make up most of the biomass on earth, Bacteria make up most of the biomass on earth, but only 1% have been culturedbut only 1% have been cultured

Classical microbiology – phenotypic approachClassical microbiology – phenotypic approach

Culture Gram stain

Shapes

Molecular microbiology – genotypic approachMolecular microbiology – genotypic approach

Identifies genesIdentifies genes

More accurate, objective, and More accurate, objective, and reproducible resultsreproducible results

Identifies unculturable bacteria (99%)Identifies unculturable bacteria (99%)

Eliminates special growth Eliminates special growth

requirementsrequirements

DNA sequence data are more DNA sequence data are more

easily shared and databasedeasily shared and databased

Rapid diagnosisRapid diagnosis

Resolves evolutionary relationshipsResolves evolutionary relationships

Genes and Genomes: fun factsGenes and Genomes: fun facts

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

Buchnera (0.64 Mb)

Blochmannia (0.75 Mb) Wolbachia (1.27 Mb)

Wigglesworthia (0.70 Mb)

Endosymbionts are the smallest bacterial genomes known

Wernegreen 2002

Insect endosymbionts

ArthropodsArthropods NematodesNematodes

Insects (20-75%!)

How do we go from here…..?How do we go from here…..?

Crustaceans (35%)

Chelicerates

2-6 million insect species are infected with

Wolbachia!!

Filarial nematodes (90%)

To studying the To studying the Wolbachia within?Wolbachia within?

Credit: Mark Taylor

PCR IntroductionPCR IntroductionPolymerase Chain Reaction (PCR) allows scientists to amplify Polymerase Chain Reaction (PCR) allows scientists to amplify minutes amount of a specific DNA sequence from a minutes amount of a specific DNA sequence from a heterogenous DNA pool in a few hoursheterogenous DNA pool in a few hours

Invented by Dr. Kary Millis in 1983 (Nobel Prize in Chemistry in Invented by Dr. Kary Millis in 1983 (Nobel Prize in Chemistry in 1993)1993)

One of the most widely used biotechnology techniques in One of the most widely used biotechnology techniques in biological research. PCR is the method of choice for symbiont biological research. PCR is the method of choice for symbiont detection.detection.

Basics of PCRBasics of PCRTemplate DNATemplate DNA - the starting - the starting DNA of interest.DNA of interest.

High temperature denatures High temperature denatures template DNA into single template DNA into single strands and synthetic strands and synthetic sequences of ssDNA (20-30 sequences of ssDNA (20-30 nucleotides) serve as nucleotides) serve as primersprimers

Two different primers are used to Two different primers are used to bracket the target gene to be bracket the target gene to be amplifiedamplified

DNA polymerase copies the DNA polymerase copies the complimentary strand starting complimentary strand starting at the primer. In one cycle, two at the primer. In one cycle, two identical strands are made.identical strands are made.

To perform your PCR - Ready BeadsTo perform your PCR - Ready Beads

Small quantity of DNASmall quantity of DNA

PrimersPrimers

Buffered solution containing Buffered solution containing DNA polymeraseDNA polymerase

Four base pairs of DNAFour base pairs of DNA

Cofactor MgClCofactor MgCl22

All in test tube

Buffered solution containing Buffered solution containing DNA polymeraseDNA polymerase

Four base pairs of DNAFour base pairs of DNA

Cofactor MgClCofactor MgCl22

Small quantity of DNASmall quantity of DNA

PrimersPrimers

Temperature drives the reactionTemperature drives the reaction

Target gene: 16S rDNATarget gene: 16S rDNA

Small ribosomal subunit involved in mRNA translation process Small ribosomal subunit involved in mRNA translation process Ancient molecule, functionally constant, universally distributedAncient 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 Present in bacterial symbionts; eukaryote has very divergent copy that is named 18S rRNAcopy that is named 18S rRNA

PCR AnimationPCR Animation

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

Two Key Innovations for Success of PCRTwo Key Innovations for Success of PCR

Heat-stable DNA polymerase Heat-stable DNA polymerase isolated from bacterium isolated from bacterium Thermus Thermus aquaticusaquaticus which inhabits hot which inhabits hot springssprings

Polymerase remains active Polymerase remains active despite being heated many timesdespite being heated many times

DNA thermal cyclers – a DNA thermal cyclers – a computer that controls repetitive computer that controls repetitive temperature changes required temperature changes required for PCRfor PCR

70C hot springs in Yellowstone National Park

Example of a thermal cycler from MJ Research

PCR and Pop CulturePCR and Pop Culture “ “Jurassic Park” and “CSI”Jurassic Park” and “CSI”

Some fun PCR facts to share with Some fun PCR facts to share with your students: …PCR has been your students: …PCR has been used to amplify DNA from…used to amplify DNA from…

a preserved quagga (a zebra a preserved quagga (a zebra relative that became extinct 100 relative that became extinct 100 years ago)years ago)crime scenes (e.g., O.J.)crime scenes (e.g., O.J.)eight-celled human preembryos, to eight-celled human preembryos, to detect cystic fibrosisdetect cystic fibrosisthe brain of a 7000 year old the brain of a 7000 year old American mummyAmerican mummypatients for disease diagnosispatients for disease diagnosis

Our goal: Determine which of your insects Our goal: Determine which of your insects harbor harbor WolbachiaWolbachia??

DNA extractionDNA extraction:: PCRPCR:: Gel electrophoresisGel electrophoresis::

Bellingham HS, MA Falmouth HS, MA Bronx HS, NY

One day till we discover the One day till we discover the microbes within!microbes within!

QuickTime™ and aPhoto - JPEG decompressor

are needed to see this picture.

2005 Workshop Participant

2007-2008

Student

The The WolbachiaWolbachia Project: A “Bridge” Project: A “Bridge” to New Science in the Classroom: to New Science in the Classroom:

PCR ResultsCross Type ID #

FlyGender Insect

DNAWolbachia

DNAPhageDNA

1 F + - -

2 F + - -

3 F + - -

6 M + - -

7 M + - -

Infected Male x

Uninfected Female

8 M + - -

11 F + + +

12 F + + +

13 F + + +

16 M + + +

17 M + + +

Infected Male x

Infected Female

18 M + + +

21 F + - -

22 F + - -

23 F + - -

26 M + - -

27 M + - -

Uninfected Male x

Uninfected Female

28 M + - -

31 F + + +

32 F + + +

33 F + + +

36 M + + +

37 M + + +

Uninfected Male x

Infected Female

38 M + + +

Notation Key(+) - positive result (-) - negative result

*Results suggest all samples have insect DNA. Results suggest samples with an infectedmother have Wolbachia and the virus. Due to an error in the dilution of the Wolbachiaprimer, research was conducted twice.

PCR ResultsCross Type ID #

FlyGender Insect

DNAWolbachia

DNAPhageDNA

1 F + - -

2 F + - -

3 F + - -

6 M + - -

7 M + - -

Infected Male x

Uninfected Female

8 M + - -

11 F + + +

12 F + + +

13 F + + +

16 M + + +

17 M + + +

Infected Male x

Infected Female

18 M + + +

21 F + - -

22 F + - -

23 F + - -

26 M + - -

27 M + - -

Uninfected Male x

Uninfected Female

28 M + - -

31 F + + +

32 F + + +

33 F + + +

36 M + + +

37 M + + +

Uninfected Male x

Infected Female

38 M + + +

Notation Key(+) - positive result (-) - negative result

*Results suggest all samples have insect DNA. Results suggest samples with an infectedmother have Wolbachia and the virus. Due to an error in the dilution of the Wolbachiaprimer, research was conducted twice.

Senior StudentNorth 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

Progression of the Wolbachia ProjectProgression of the Wolbachia Project

ProfessionalDevelopmentProfessionalDevelopment

New ScienceNew Science

ImplementationImplementation

Summer Envisionships

Summer Envisionships