Lecture 5: Biotechnology and prokaryotes

1. Lecture 5: Biotechnology and prokaryotes • Bacteria and bacterial systems; • Manipulation of gene expression in prokaryotes (Chapt 6); • Large-scale purification of proteins from • recombinant microorganisms (Chapt 16)

2. Biotechnology and prokaryotes • “germs” • Prokaryotes are ubiquitous • Wide spectrum of species, types, characteristics • “Relatively simple” systems, for manipulation • Biology characterized • Genetics characterized • [Molecular biology characterized] • Genomics, bioinformatics, systems • Industrialization, [weapon-ization] • Two-edged blade: “anti- and pro-” • Effects on humans, other organisms, environment • [medical (pathogen), therapeutic (food)]… • Less legal, ethical, moral issues

3. Phylogeny and range of prokaryotes

4. Phylogenic branches • TThiel, UMissouri • www.umsl.edu/~microbes/pdf/introductiontobacteria.pdf

5. Shapes and “multicellular”-ism • quorum • fluorescence • biofilms • ‘differentiation’- Myxococcus xanthus • TThiel, UMissouri • www.umsl.edu/~microbes/pdf/introductiontobacteria.pdf

6. Optimum growth temperature • TThiel, UMissouri • www.umsl.edu/~microbes/pdf/introductiontobacteria.pdf

7. Structure of a type of bacterium

8. Types of bacteria: gram positive vs gram negative • wikipedia • Characterization of bacteria: Gram stain • Hans Christian Gram, 1882 • Crystal violet -> methylene blue

9. Types of bacteria: gram positive vs gram negative wikipedia • Gram positive: • Thick cell wall, peptidoglycan • Examples: Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus, Clostridium • Gram negative: • Cell wall, lipopolysaccharide (also, LPS or endotoxin layer) plus [less] peptidoglycan • Examples: Escherichia coli, Salmonella, Pseudomonas, Legionella

10. Bacillus types and biotechnology • Gram positive: • Contrast B. anthracis vs. B. thuringiensis vs B. subtilis • Spores vs crystals • Ag biotech • http://www.scq.ubc.ca/wp-content/uploads/2006/08/GM-crop.gif • http://images.google.com/imgres?imgurl=http://www.magma.ca/~scimat/B_thur16.jpg&imgrefurl=http://www.magma.ca/~scimat/ • B_thurin.htm&h=324&w=340&sz=23&tbnid=4A1w35t4YFto8M:&tbnh=113&tbnw=119&prev=/images%3Fq%3Dbacillus%2Bthuringiensis • %26um%3D1&start=1&sa=X&oi=images&ct=image&cd=1 • wikipedia

11. Bacillus and biotechnology • Gram positive: • B. thuringiensis • Spores vs crystals • Ag biotech • http://www.scq.ubc.ca/wp-content/uploads/2006/08/GM-crop.gif • http://images.google.com/imgres?imgurl=http://www.magma.ca/~scimat/B_thur16.jpg&imgrefurl=http://www.magma.ca/~scimat/ • B_thurin.htm&h=324&w=340&sz=23&tbnid=4A1w35t4YFto8M:&tbnh=113&tbnw=119&prev=/images%3Fq%3Dbacillus%2Bthuringiensis • %26um%3D1&start=1&sa=X&oi=images&ct=image&cd=1 • wikipedia

12. Wall-less bacteria • [http://bacteriality.com/2007/08/18/history/] • Wall-less bacteria • Smallest, simplest free-living self-replicating organisms; still, complex • Reduced genome species • ~100 species of bacteria and archaea • Membrane ~30% lipid and 70% protein • 25-30% cholesterol, comparable to eukaryotes • [other bacteria do not have cholesterol or sterols in PM] • Example: Mycoplasma pneumoniae, Thermoplasma acidophilum • L-form or cell wall deficient bacteria • Chronic disease? [rheumatoid arthritis, Chronic Fatigue Syndrome, Lyme disease]

13. Life on the Edge • Deinococcus radiodurans (eubacteria) • Survives 5 Mrads • DNA highly damaged at 1.7 Mrads; 1,000 to 2,000 DNA fgmts repaired in 24 hrs • RE map; Four genome elements, circular <-> linear large http://www.ornl.gov/sci/techresources/Human_Genome/publicat/microbial/image3.html

14. Life on the Edge • Science Daily (Source: NASA, +), 4/4/00 “Weird Life” • The limits of life on Earth are much broader than previously thought. • Examples of life at extreme conditions include: • Highest radiation: 5 million rads-- Deinococcus radiodurans (eubacteria) • Hottest: 235.4 F (113 C)-- bacteria from deep sea vents • Coldest: 5 F (-15 C)-- microalgae in Antarctic rocks • Deepest: Two miles underground in rocks-- bacteria • Most acidic: pH 0-- Unclassified organisms growing on gypsum in caves • Basic: pH 9.0; 12% salt-- bacteria, Soap Lake, Southeast WA • Saltiest: 30 percent salt environment-- bacteria • Deepest and Highest pressure: 1200 atm-- at bottom of Marianas Trench (ocean) • [Farthest: Moon-- Streptococcus mitus (from human source) from Surveyor III camera after three • years unprotected on lunar surface]

15. Varied growth rates, oxygen needs • 20 min: E. coli • 24 hrs: M. tuberculosis • TThiel, UMissouri • www.umsl.edu/~microbes/pdf/introductiontobacteria.pdf

16. Manipulating bacteria • http://www.mansfield.ohio-state.edu/~sabedon/black06.htm

17. Bacterial growth • http://www-micro.msb.le.ac.uk/labwork/bact/bact1.htm

18. Bacterial growth: quantify

19. Natural selection and growth • Thomas Malthus. 1798, “Essay on the Principle of Population” • “population would outstrip food supply” • Charles Darwin. 1859, “The Origin of Species” • “an application of doctrines of Malthus”

20. Natural selection and growth: How? • Glucose- depleted • Fructose- not available • Maltose- not available • Lactose- available

21. Microbial genetics and molecular biology • Genes and pathways are under strict regulatory control

22. Microbial genetics and molecular biotechnology • Control timing of expression of foreign gene product • Lac promoter • Repressed by lac repressor • Derepressed by lactose and IPTG • lacUV5 -nuc change in -10 region • Stronger promoter • Trp promoter • Repressed by trp repressor • Derepressed by trp and indoleacrylic acid • “leaky”

23. hybrid promoter: tac • Overexpression of foreign gene product • eg, lac repressor protein: • ~10 molecules per cell • ‘need’ ~1 umol for biochem studies • 1 umol = 6 x1017 molecules • Or 6 x1016 cells • E. coli at stationary phase has • ~4 x108 cells/mL • *if* 100% recovery, need 150,000 L • tac promoter • Hybrid of trp and lac promoters • Matches consensus for E. coli RNAP seq • HAdeBoer, et al. 83. PNAS 80:21

24. hybrid promoter: tac [not always straight-forward] • tac promoter • tacI, tacII • lacUV5 -nuc change in -10 region • Relative strengths • tac/lac 11.8x; tac/trp 3.5x • Strain dependent • HAdeBoer, et al. 83. PNAS 80:21

25. Bacteriophage genetics

26. Bacteriophage genetics and molecular biology • Lambda promoter • Temperature sensitive promoter • Repressed unless temperature raised • Overexpression of foreign gene product • incompatible or lethal/deleterous

27. dual promoters: lac and T7 • Repressed unless inducer is present • Overexpression of foreign gene product • incompatible or lethal/deleterous

28. Plasmids • JLederberg, 1952. Physiol. Rev. 32: 403 -”plasmid” • Circular, extrachromosomal double-stranded DNA • Size: 1 kb to 400 kb • Number of copies: 1-2 to 20s to 100s ~ori • wikipedia

29. Plasmids • Conjugation- horizontal gene transfer • vs ‘episome’ (plasmid that can integrate into chromosomal DNA) • Size: 1 kb to 400 kb • Number: 1-2 to 20s to 100s ~ori wikipedia

30. Overexpression strategy: construction of hybrid vector • Have: pPLc2833 • Strong promoter pL, selectable marker, MCS • Have: pKN402 ori gives increased copy number 5-10x • Temperature-dependent copy number • Construct: hybrid pCP3 • pL promoter and ApR gene with high-copy ori

31. Overexpression strategy: plasmid number increase • Have: pPLc2833 • Strong promoter pL, selectable marker, MCS • Have: pKN402 ori gives increased copy number 5-10x • Temperature-dependent copy number • Construct: hybrid pCP3 • pL promoter and ApR gene with high-copy ori

32. Large-scale prep considerations • IPTG and other derepressing chemicals can become expensive • Temperature-dependent shifts: time and energy for large cultures • Reconfigure pL with trp promoter • Grow in molasses and casein hydrolysate, low in free trp; derepress with tryptone [crude extract] • ex, -galactosidase and citrate synthase genes-> overproduced to 21% and 24% of cellular proteins

33. Other bacterial hosts • Genetics and molecular biology not as well-developed as E. coli • Using E. coli system in other gram negatives • (Nm is neomycin resistance; S1 is R. meliloti ribo protein gene)

34. Universal gram negative vector • 70 bp fgmt from Tn-5 Terminal IR • Low copy-number broad-host-range plasmid • E. coli, Alcaligenes sp, Enterobacter cloacae, Klebsiella pneumoniae, • Pseudomonas stutzeri, Pseudomonas fluorescens, Serratia marcescens

35. Modifications for human consumption • Lactic acid metabolizing bacteria, eg Lactococcus spp • Dairy products- cheese and yogurt • Desirable to increase yields, add to quality of food • Undesirable to alter production process, product palatability, appearance, etc. • Cannot add chemical inducers or temperature effects to processing • Consensus constitutive promoter--> synthetic constitutive promoters • Tested 36 constructs • Most active ~7,000x stronger than least active • If -10 or -35 regions intact, ~400x in strength

36. Fusion proteins • Not all heterologously expressed proteins are expressed • Not all heterologously expressed proteins are expressed ‘enough’ • Not all heterologously expressed proteins are stable as expressed • Engineer a fusion protein that has a partial structure of a ‘host-native’ • Increase expression • Extend half-life • Requires correct reading frame • Above, proteolytic cleavage by blood coagulation factor Xa • Val is N-term of desired protein • Based on existing biology….

37. Blood clotting

38. Post-translational modifications • Extend half-life • Address protein delivery • Stabilize structure • Enhance biological activity

39. Vector for expression fused protein • 5’-terminal segment of ompF gene for producing antigen • Directs synthesis of outer membrane protein and part of -galactosidase • Omp provides signals for txn and tnl, and secretion of product • lacZ functional, as a reporter for in-frame • “tribrid” has all three products’ characteristics

40. Purification protocol enhancement • Efficient protocols for purification of product

41. Fusion construct example • Product is a secretion fusion protein • Fusion is marker peptide plus interleukin-2 • Marker protein serves to extend half-life and to enable rapid purification • One-step purification via immunoaffinity chromatography • Small marker peptide does not stress host resources • For FDA, removal specifically with bovine intestinal enterokinase, a protease

42. Fusion protein purification

43. Column chromatography/HPLC

44. “Recombinant DNA era” protein purification • DNFrick and MJBessman JBC 270: 1529 (95) • Over-expression and purification of the Orf257 protein: SDS-PAGE and PAGE • A1) markers; A2 and 3) -/+IPTG;A 4 and 5) 2 and 6 ug Fraction V • B1) markers; B2 and 3) 2 and 6 ug Fraction V • Protocol: • 1. DEAE-Sepharose • 2. Streptomycin-SO4 fractionation • 3. Sephadex G-100 chromatography

45. Rapid screening methods: bacteriophage • Large libraries of recombinants- How to find clone of interest? • ex., cDNA library with 5 x1010 clones, proteins which are rarely occurring cDNAs • Can fuse with a surface protein gene of either a filamentous phage or bacterium • After txn and tnl, fusion is expressed on the surface of the organism • Can screen with antibody

46. Rapid screening methods: bacterium • Large libraries of recombinants- How to find clone of interest? • ex., cDNA library with 5 x1010 clones, proteins which are rarely occurring cDNAs • Can fuse with a surface protein gene of either a filamentous phage or bacterium • After txn and tnl, fusion is expressed on the surface of the organism • Can screen with antibody

47. Using probes: practice, alternatives and updates (surface display)

48. Overexpression of foreign product • Increasing plasmid copy number sometimes reduces yield of product • Cell resources diverted to production of other plasmid-encoded products • Creation of tandem arrays of the product • In-frame expression of multiple copies • Each copy has own signals • Copy number limits yield as well; unstable inserts

49. Translation expression vectors • Requires more than regulated strong promoter • Efficiency of translation and stability of nascent peptide • Prokaryotes: proteins are synthesized at different efficiencies, up to 100x • One aspect- translational initiation signal or ribosome-binding site • If mRNA has hairpin, can disrupt • Above GGGGG is rbs and AUG is start

50. Construction of a translation efficient vector • ApR marker • tac promoter • lacZ ribosome-binding site • ATG start site, 8 nucs downstream from rbs • Two txn terminators T1 and T2 from lambda