1. Engineering magnetosomes to express novel proteins Which ones? Must be suitable for expressing in Magnetospyrillum! Can’t rely on glycosylation, disulphide bonds, lipidation, selective proteolysis, etc for function! Best bets are bacterial proteins Alternatives are eukaryotic proteins that don’t need any of the above Short peptides Tweaking p18 Linker Deleting or replacing GFP TRZN Oxalate decarboxylases Lactate dehydrogenase or other oxalate metab enzyme

2. How to bioengineer a novel protein expression system? 1.Identify a suitable candidate organism 2.DNA must function in this host 3.mRNA must function in this host 4.Protein must be functional Must be able to purify large amounts of functional protein

3. How to bioengineer a novel protein expression system New host must be able to “read” the sequence Promoters Terminators Capping, splicing and polyA if eukaryote Codon usage

4. How to bioengineer a novel protein expression system New host must be able to “read” the sequence Promoters Terminators Capping, splicing and polyA if eukaryote Codon usage 3. mRNA must function in the new host

5. How to bioengineer a novel protein expression system? 3. mRNA must function in the new host Must be translated: needs correct ribosome-binding site

6. How to bioengineer a novel protein expression system? 3. mRNA must function in the new host Must be translated: needs correct ribosome-binding site S-D in prok Kozak in euk

7. How to bioengineer a novel protein expression system? 3. mRNA must function in the new host Must be translated: needs correct ribosome-binding site S-D in prok Kozak in euk Also needs correct information in 5’ UTR

8. How to bioengineer a novel protein expression system? 3. mRNA must function in the new host Must be translated: needs correct ribosome-binding site Also needs correct information in 5’ UTR In eukaryotes also needs 5’ cap & poly-A tail

9. How to bioengineer a novel protein expression system? 3. mRNA must function in the new host Must be translated: needs correct ribosome-binding site Also needs correct information in 5’ UTR In eukaryotes also needs 5’ cap & poly-A tail Must also be properly localized

10. How to bioengineer a novel protein expression system? 3. mRNA must function in the new host Must be translated: needs correct ribosome-binding site Must survive: in euk depends on internal sequences (especially in 3’ UTR) and poly-A tail

11. How to bioengineer a novel protein expression system? 4. Protein must function in the new host Must be translated Must be modified correctly

12. How to bioengineer a novel protein expression system? 4. Protein must function in the new host Must be translated Must be modified correctly Glycosylation: affects folding, solubility & localization

13. How to bioengineer a novel protein expression system? 4. Protein must function in the new host Must be translated Must be modified correctly Glycosylation: affects folding, solubility & localization Simple glycosylation can be engineered

14. Protein must function in the new host Must be modified correctly Glycosylation Lipidation Myristylation (14C) @ N Palmitoylation (16C) Isoprenoids

15. Protein must function in the new host Must be modified correctly Glycosylation Lipidation proteolysis

16. Protein must function in the new host Must be modified correctly Glycosylation Lipidation Proteolysis Disulfide bonds

17. Protein must function in the new host Must be modified correctly Glycosylation Lipidation Proteolysis Disulfide bonds Must go to correct location

18. Protein must function in the new host Must be modified correctly Glycosylation Lipidation proteolysis Must go to correct location Must survive!

19. How to bioengineer a novel protein expression system? 3. Protein must function in the new host Must be modified correctly Glycosylation Lipidation proteolysis Must go to correct location Must survive! 4. Identify suitable gene(s), then obtain in some way, add suitable promoters and terminators, and transform into new host.

20. How to bioengineer a novel protein expression system? Hosts? Escherichia coli Pro Best-understood & fastest growing of all hosts Most genetic control Lots of tricks & special-purpose strains

21. How to bioengineer a novel protein expression system? Hosts? Escherichia coli Pro Best-understood & fastest growing of all hosts Most genetic control Lots of tricks & special-purpose strains Con Prokaryote: no glycosylation, S-S bonds, etc.

22. How to bioengineer a novel protein expression system? Hosts? Escherichia coli Pro Best-understood & fastest growing of all hosts Most genetic control Lots of tricks & special-purpose strains Con Prokaryote: no glycosylation, S-S bonds, etc. Saccharomyces cerevisiae (brewer’s yeast) Pro Eukaryotic Fast-growing and well-characterized Good genetic control Lots of tricks & special-purpose strains

23. Hosts? E. coli S. cerevisiae (brewer’s yeast) Pro Eukaryotic Fast-growing and well-characterized Good genetic control Lots of tricks & special-purpose strains Con Does things its own way Weird glycosylation, etc Proteins from other eukaryotes often don’t work when expressed in yeast

24. Hosts? E. coli Saccharomyces cerevisiae (brewer’s yeast) Pichia pastoris Pro Methylotrophic: grows in [methanol] that kill most other organisms Eukaryotic Fast-growing and well-characterized Good genetic control with glucose & MeOH Grows to very high densities

25. Hosts? E. coli Saccharomyces cerevisiae (brewer’s yeast) Pichia pastoris Pro Methylotrophic: grows in [methanol] that kill most other organisms Eukaryotic Fast-growing and well-characterized Good genetic control with glucose & MeOH Grows to very high densities Con Does things its own way Weird glycosylation, etc Proteins from other euk often don’t work

26. Hosts? E. coli Saccharomyces cerevisiae (brewer’s yeast) Pichia pastoris Baculovirus in cultured insect cells Pro Make glycoproteins “faithfully” Can’t infect plants or animals Can use promoters of “late genes” to drive expression of heterologous proteins Insect cell cultures are easier & more forgiving than mammalian cell cultures

27. Hosts? E. coli Saccharomyces cerevisiae (brewer’s yeast) Pichia pastoris Baculovirus in cultured insect cells Pro Make glycoproteins “faithfully” Can’t infect plants or animals Can use promoters of “late genes” to drive expression of heterologous proteins Insect cell cultures are easier & more forgiving than mammalian cell cultures Con Trickier Requires cell cultures: expensive!

28. Hosts? E. coli Saccharomyces cerevisiae (brewer’s yeast) Pichia pastoris Baculovirus in cultured insect cells Transgenic plants Pro Make authentic plant proteins (and sometimes animal proteins) Cheap “Easy” and robust techniques

29. Hosts? E. coli Saccharomyces cerevisiae (brewer’s yeast) Pichia pastoris Baculovirus in cultured insect cells Transgenic plants Pro Make authentic plant proteins (and sometimes animal proteins) Cheap “Easy” and robust techniques Con Slow May not process non-plant proteins correctly

30. Hosts? E. coli Saccharomyces cerevisiae (brewer’s yeast) Pichia pastoris Baculovirus in cultured insect cells Transgenic plants Animal cell cultures Pro Make authentic animal proteins “Easy” and robust techniques

31. Hosts? E. coli Saccharomyces cerevisiae (brewer’s yeast) Pichia pastoris Baculovirus in cultured insect cells Transgenic plants Animal cell cultures Pro Make authentic animal proteins “Easy” and robust techniques Con Slow Expensive! Purifying proteins can be difficult

32. Hosts? E. coli Saccharomyces cerevisiae (brewer’s yeast) Pichia pastoris Baculovirus in cultured insect cells Transgenic plants Animal cell cultures Transgenic animals Pro Make authentic animal proteins

33. Hosts? E. coli Saccharomyces cerevisiae (brewer’s yeast) Pichia pastoris Baculovirus in cultured insect cells Transgenic plants Animal cell cultures Transgenic animals Pro Make authentic animal proteins Con Slow Expensive! Difficult Purifying proteins can be difficult

34. Hosts? Magnetospirillum gryphiswaldense

35. Hosts? Magnetospirillum gryphiswaldense Can propagate plasmids (but pBAM requires pir gene) Or can insert into chromosome via tnpA (Tn5)-based transposition: no variation in expression due to copy number or growth stage

36. Hosts? Magnetospirillum gryphiswaldense Borg optimised rbs, promoter & codon usage

37. Hosts? Magnetospirillum gryphiswaldense Borg optimised rbs, promoter & codon usage Developed inducible system based on tetracycline

38. Hosts? Magnetospirillum gryphiswaldense Borg optimised rbs, promoter & codon usage Developed inducible system based on tetracycline Fuse protein to C-terminus of mamC

39. Hosts? Magnetospirillum gryphiswaldense Borg optimised rbs, promoter & codon usage Developed inducible system based on tetracycline Fuse protein to mamC C-terminus: exposed at surface

40. Hosts? Magnetospirillum gryphiswaldense Borg optimised rbs, promoter & codon usage Developed inducible system based on tetracycline Fuse protein to mamC C-terminus: exposed at surface Purify with magnets

41. Assignment Design a mamC C-terminal protein fusion Design DNA sequence encoding a useful protein

42. Assignment Design a mamC C-terminal protein fusion Design DNA sequence encoding a useful protein Replace eGFP of pJH3 with your protein Best to use MluI and NheI sites

43. Assignment Best to use MluI and NheI sites  Design oligos that add MluI in frame at 5” end and NheI at 3’end

44. Assignment Best to use MluI and NheI sites  Design oligos that add MluI in frame at 5’ and NheI at 3’end  Digest vector & clone with MluI and NheI then ligate

45. Assignment Best to use MluI and NheI sites  Design oligos that add MluI in frame at 5’ and NheI at 3’end  Digest vector & clone with MluI and NheI then ligate  Find & analyze clones

46. Transcription Prokaryotes have one RNA polymerase makes all RNA core polymerase = complex of 5 subunits (      ’  )

47. Transcription Prokaryotes have one RNA polymerase makes all RNA core polymerase = complex of 5 subunits (      ’  )  not absolutely needed, but cells lacking  are very sick

48. Initiating transcription in Prokaryotes 1) Core RNA polymerase is promiscuous

49. Initiating transcription in Prokaryotes 1)Core RNA polymerase is promiscuous 2)sigma factors provide specificity

50. Initiating transcription in Prokaryotes 1)Core RNA polymerase is promiscuous 2)sigma factors provide specificity Bind promoters

51. Initiating transcription in Prokaryotes 1)Core RNA polymerase is promiscuous 2)sigma factors provide specificity Bind promoters Different sigmas bind different promoters

52. Initiating transcription in Prokaryotes 1)Core RNA polymerase is promiscuous 2)sigma factors provide specificity Bind promoters 3) Once bound, RNA polymerase “melts” the DNA

53. Initiating transcription in Prokaryotes 3) Once bound, RNA polymerase “melts” the DNA 4) rNTPs bind template

54. Initiating transcription in Prokaryotes 3) Once bound, RNA polymerase “melts” the DNA 4) rNTPs bind template 5) RNA polymerase catalyzes phosphodiester bonds, melts and unwinds template

55. Initiating transcription in Prokaryotes 3) Once bound, RNA polymerase “melts” the DNA 4) rNTPs bind template 5) RNA polymerase catalyzes phosphodiester bonds, melts and unwinds template 6) sigma falls off after ~10 bases are added

56. Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5’ to transcription start) 5’-TATAAT-3’ determines exact start site: bound by  factor

57. Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5 ’ to transcription start) 5 ’ -TATAAT-3 ’ determines exact start site: bound by  factor 2) ” -35 region ” : 5 ’ -TTGACA-3 ’ : bound by  factor

58. Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5 ’ to transcription start) 5 ’ -TATAAT-3 ’ determines exact start site: bound by  factor 2) ” -35 region ” : 5 ’ -TTGACA-3 ’ : bound by  factor 3) UP element : -57: bound by  factor

59. Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5 ’ to transcription start) 5 ’ -TATAAT-3 ’ determines exact start site: bound by  factor 2) ” -35 region ” : 5 ’ -TTGACA-3 ’ : bound by  factor 3) UP element : -57: bound by  factor

60. Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5 ’ to transcription start) 5 ’ -TATAAT-3 ’ determines exact start site: bound by  factor 2) ” -35 region ” : 5 ’ -TTGACA-3 ’ : bound by  factor 3) UP element : -57: bound by  factor Other sequences also often influence transcription! Eg CAP site in lac promoter

61. Termination of transcription in prokaryotes 1) Sometimes go until ribosomes fall too far behind

62. Termination of transcription in prokaryotes 1) Sometimes go until ribosomes fall too far behind 2) ~50% of E.coli genes require a termination factor called “rho”

63. Termination of transcription in prokaryotes 1) Sometimes go until ribosomes fall too far behind 2) ~50% of E.coli genes require a termination factor called “rho” 3) Our terminator (rrnB) first forms an RNA hairpin, followed by an 8 base sequence TATCTGTT that halts transcription