1. BEGR 424 Molecular Biology William Terzaghi Spring, 2016

2. BEGR424- Resource and Policy Information Instructor: Dr. William Terzaghi Office: SLC 363/CSC228 Office hours: MWF 12:00-1:00, TR 1-2 or by appointment Phone: (570) 408-4762 Email: terzaghi@wilkes.edu

3. BEGR424- Resource and Policy Information Instructor: Dr. William Terzaghi Office: SLC 363/CSC228 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/BEGR424.htm l

4. General considerations What do you hope to learn?

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

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

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

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

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

10. Rather than following a set series of lectures, study a problem and see where it leads us.

11. 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

12. 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

13. 1.Pick a problem

14. 2.Design some experiments

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

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

17. 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?

18. 1.Trying to find another way to remove oxalate

19. 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?

20. 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 pO 2

21. 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 pO 2 Can engineer Mms13-fusion proteins

22. 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 ncRNA Making Crispr/CAS9 proteins Mutate/replace specific genes Bind specific DNA sequences Color code with fluorescent proteins Repress expression Make transcriptional activators by fusing with activation domains

23. 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 ncRNA 5. Studying sugar signaling

24. 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 ncRNA 5. Studying sugar signaling 6. Bioremediation Atrazine Neonicotinoid pesticides Something else??

25. 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 ncRNA 5. Studying sugar signaling 6. Bioremediation 7.Making plants/algae that bypass Rubisco to fix CO 2

26. 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 ncRNA 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???

27. 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 ncRNA 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?

28. 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

29. Genome Projects Studying structure & function of genomes

30. Genome Projects Studying structure & function of genomes Sequence first

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

32. Genome Projects How much DNA is there? SV40 has 5000 base pairs E. coli has 5 x 10 6 Yeast has 2 x 10 7 Arabidopsis has 10 8 Rice has 5 x 10 8 Humans have 3 x 10 9 Soybeans have 3 x 10 9 Toads have 3 x 10 9 Salamanders have 8 x 10 10 Lilies have 10 11

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

34. Genome Projects C-value paradox: DNA content/haploid genome varies widely Some phyla show little variation: birds all have ~10 9 bp

35. Genome Projects C-value paradox: DNA content/haploid genome varies widely Some phyla show little variation: birds all have ~10 9 bp mammals all have ~ 3 x 10 9 bp

36. Genome Projects C-value paradox: DNA content/haploid genome varies widely Some phyla show little variation: birds all have ~10 9 bp mammals all have ~ 3 x 10 9 bp Other phyla are all over: insects and amphibians vary 100 x

37. Genome Projects C-value paradox: DNA content/haploid genome varies widely Some phyla show little variation: birds all have ~10 9 bp mammals all have ~ 3 x 10 9 bp Other phyla are all over: insects and amphibians vary 100 x flowering plants vary 1000x

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

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

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

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

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

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

44. Cot curves denature (melt) DNA by heating

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

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

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

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

49. 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

50. 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]

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

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

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

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

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