BEGR 424 Molecular Biology William Terzaghi Spring 2016

BEGR 424 Molecular Biology William Terzaghi Spring, 2016

BEGR 424 - Resource and Policy Information Instructor: Dr. William Terzaghi Office: SLC 363/CSC 228 Office hours: MWF 12: 00 -1: 00, TR 1 -2 or by appointment Phone: (570) 408 -4762 Email: terzaghi@wilkes. edu

BEGR 424 - Resource and Policy Information Instructor: Dr. William Terzaghi Office: SLC 363/CSC 228 Office hours: MWF 12: 00 -1: 00, TR 1 -2 or by appointment Phone: (570) 408 -4762 Email: terzaghi@wilkes. edu Course webpage: http: //staffweb. wilkes. edu/william. terzaghi/BEGR 424. html

General considerations What do you hope to learn?

General considerations What do you hope to learn? Graduate courses 1. learning about current literature

General considerations What do you hope to learn? Graduate courses 1. learning about current literature • Learning how to give presentations

General considerations What do you hope to learn? Graduate courses 1. learning about current literature 2. Learning current techniques

General considerations What do you hope to learn? Graduate courses 1. learning about current literature 2. Learning current techniques • Using them!

Provide a genuine experience in using cell and molecular biology to learn about a fundamental problem in biology.

Provide a genuine experience in using cell and molecular biology to learn about a fundamental problem in biology. • Rather than following a set series of lectures, study a problem and see where it leads us.

Provide a genuine experience in using cell and molecular biology to learn about a fundamental problem in biology. • Rather than following a set series of lectures, study a problem and see where it leads us. • Lectures & presentations will relate to current status

Provide a genuine experience in using cell and molecular biology to learn about a fundamental problem in biology. • Rather than following a set series of lectures, study a problem and see where it leads us. • Lectures & presentations will relate to current status • Some class time will be spent in lab & vice-versa • we may need to come in at other times as well

1. Pick a problem

1. Pick a problem 2. Design some experiments

1. Pick a problem 2. Design some experiments 3. See where they lead us

1. Pick a problem 2. Design some experiments 3. See where they lead us Grading? Combination of papers and presentations

GRADING? Combination of papers and presentations • First presentation: 5 points • Research presentation: 10 points • Final presentation: 15 points • Assignments: 5 points each • Poster: 10 points • Intermediate report 10 points • Final report: 30 points Alternatives • Paper(s) instead of 1 or two presentations • Research proposal instead of a presentation • One or two exams?

1. Trying to find another way to remove oxalate

Topics? 1. Trying to find another way to remove oxalate 2. Making a probiotic bacterium that removes oxalate • Identifying best candidates • Figuring out how to engineer them • Add oxalate transporter? • Add more/different oxalate altering enzymes? • Target them to different locations?

Topics? 1. Trying to find another way to remove oxalate 2. Making a probiotic bacterium that removes oxalate 3. Engineering magnetosomes to express novel proteins • Membrane-bound single-domain iron-oxide crystals made by magnetotic bacteria to help find correct p. O 2

Topics? 1. Trying to find another way to remove oxalate 2. Making a probiotic bacterium that removes oxalate 3. Engineering magnetosomes to express novel proteins • Membrane-bound single-domain iron-oxide crystals made by magnetotic bacteria to help find correct p. O 2 • Can engineer Mms 13 -fusion proteins

Topics? 1. Trying to find another way to remove oxalate 2. Making a probiotic bacterium that removes oxalate 3. Engineering magnetosomes to express novel proteins 4. Studying nc. RNA • Making Crispr/CAS 9 proteins • Mutate/replace specific genes • Bind specific DNA sequences • Color code with fluorescent proteins • Repress expression • Make transcriptional activators by fusing with activation domains

Topics? 1. Trying to find another way to remove oxalate 2. Making a probiotic bacterium that removes oxalate 3. Engineering magnetosomes to express novel proteins 4. Studying nc. RNA 5. Studying sugar signaling

Topics? 1. Trying to find another way to remove oxalate 2. Making a probiotic bacterium that removes oxalate 3. Engineering magnetosomes to express novel proteins 4. Studying nc. RNA 5. Studying sugar signaling 6. Bioremediation • Atrazine • Neonicotinoid pesticides • Something else? ?

Topics? 1. Trying to find another way to remove oxalate 2. Making a probiotic bacterium that removes oxalate 3. Engineering magnetosomes to express novel proteins 4. Studying nc. RNA 5. Studying sugar signaling 6. Bioremediation 7. Making plants/algae that bypass Rubisco to fix CO 2

Topics? 1. Trying to find another way to remove oxalate 2. Making a probiotic bacterium that removes oxalate 3. Engineering magnetosomes to express novel proteins 4. Studying nc. RNA 5. Studying sugar signaling 6. Bioremediation 7. Making plants/algae that bypass Rubisco to fix CO 2 8. Making novel biofuels • blue-green algae that generate electricity • Plants/algae that make methane or hydrogen • Biodiesel • Other ideas? ? ?

Topics? 1. Trying to find another way to remove oxalate 2. Making a probiotic bacterium that removes oxalate 3. Engineering magnetosomes to express novel proteins 4. Studying nc. RNA 5. Studying sugar signaling 6. Bioremediation 7. Making plants/algae that bypass Rubisco to fix CO 2 8. Making novel biofuels 9. Making vectors for Dr. Harms 10. Something else?

Assignments? 1. identify a gene and design primers 2. presentation on new sequencing tech 3. designing a protocol to verify your clone 4. presentations on gene regulation 5. presentation on applying mol bio Other work 1. draft of report on cloning & sequencing 2. poster for symposium 3. final gene report 4. draft of formal report 5. formal report

Genome Projects Studying structure & function of genomes

Genome Projects Studying structure & function of genomes • Sequence first

Genome Projects Studying structure & function of genomes • Sequence first • Then location and function of every part

Genome Projects How much DNA is there? SV 40 has 5000 base pairs E. coli has 5 x 106 Yeast has 2 x 107 Arabidopsis has 108 Rice has 5 x 108 Humans have 3 x 109 Soybeans have 3 x 109 Toads have 3 x 109 Salamanders have 8 x 1010 Lilies have 1011

Genome Projects C-value paradox: DNA content/haploid genome varies widely

Genome Projects C-value paradox: DNA content/haploid genome varies widely Some phyla show little variation: birds all have ~109 bp

Genome Projects C-value paradox: DNA content/haploid genome varies widely Some phyla show little variation: birds all have ~109 bp mammals all have ~ 3 x 109 bp

Genome Projects C-value paradox: DNA content/haploid genome varies widely Some phyla show little variation: birds all have ~109 bp mammals all have ~ 3 x 109 bp Other phyla are all over: insects and amphibians vary 100 x

Genome Projects C-value paradox: DNA content/haploid genome varies widely Some phyla show little variation: birds all have ~109 bp mammals all have ~ 3 x 109 bp Other phyla are all over: insects and amphibians vary 100 x flowering plants vary 1000 x

C-value paradox One cause = variations in chromosome numbers and ploidy 2 C chromosome numbers vary widely Haplopappus has 2

C-value paradox One cause = variations in chromosome numbers and ploidy 2 C chromosome numbers vary widely Haplopappus has 2 Arabidopsis has 10

C-value paradox One cause = variations in chromosome numbers and ploidy 2 C chromosome numbers vary widely Haplopappus has 2 Arabidopsis has 10 Rice has 24 Humans have 46 Tobacco (hexaploid) has 72 Kiwifruit (octaploid) have 196

C-value paradox Chromosome numbers vary So does chromosome size!

C-value paradox Chromosome numbers vary So does chromosome size! Reason = variation in amounts of repetitive DNA

C-value paradox Chromosome numbers vary So does chromosome size! Reason = variation in amounts of repetitive DNA first demonstrated using Cot curves

Cot curves • denature (melt) DNA by heating

Cot curves • denature (melt) DNA by heating dissociates into two single strands

Cot curves 1. denature (melt) DNA by heating 2. Cool DNA

Cot curves 1. denature (melt) DNA by heating 2. Cool DNA: complementary strands find each other & anneal

Cot curves 1. denature (melt) DNA by heating 2. Cool DNA: complementary strands find each other & anneal • hybridize

Cot curves 1. denature (melt) DNA by heating 2. Cool DNA: complementary strands find each other & anneal • Hybridize: don't have to be the same strands

Cot curves 1. denature (melt) DNA by heating 2. Cool DNA: complementary strands find each other & anneal • Hybridize: don't have to be the same strands 3. Rate depends on [complementary strands]

Cot curves 1) denature DNA 2) cool DNA 3) at intervals measure [single-stranded DNA]

Cot curves viruses & bacteria show simple curves Cot is inversely proportional to genome size

Cot curves eucaryotes show 3 step curves Step 1 renatures rapidly: “highly repetitive”

Cot curves eucaryotes show 3 step curves Step 1 renatures rapidly: “highly repetitive” Step 2 is intermediate: “moderately repetitive”

Cot curves eucaryotes show 3 step curves Step 1 renatures rapidly: “highly repetitive” Step 2 is intermediate: “moderately repetitive” Step 3 is ”unique"