1 / 34

Gene Expression Systems in Prokaryotes

Gene Expression Systems in Prokaryotes. In Escherichia coli - lambda system - p L - T7 system – p T7 - Lac system - p lac - Trp system - synthetic systems – p tac , p trc - controlled by recombinase In Bacillus + other bacteria. E. coli Promoter Sites.

malo
Download Presentation

Gene Expression Systems in Prokaryotes

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Gene Expression Systems in Prokaryotes • In Escherichia coli - lambda system - pL - T7 system – pT7 - Lac system - plac - Trp system - synthetic systems – ptac, ptrc - controlled by recombinase • In Bacillus + other bacteria

  2. E. coli Promoter Sites

  3. E. coli Promoter Sites

  4. E. coli Promoter Sites

  5. Lambda promoter system Constitutive

  6. The T7 promoter system

  7. The T7 promoter system

  8. The T7 promoter system

  9. The T7 promoter system

  10. The Lac promoter System

  11. The trp promoter system

  12. Synthetic E. coli promoters -35 -10 Hybrid Promoters: ptac Promoter (Ptrp –35 +16pb + Plac –10; IPTG) ptrc Promoter (Ptrp –35 +17pb + Plac –10; IPTG)-> invitro most powerful -> invivo 90% of ptac ptic Promoter (Ptrp –35 +18pb + Plac –10; IPTG) -> 65 % of ptac

  13. Synthetic E. coli promoters

  14. Phase Switch Promoter System (from Salmonella) -> for very toxic proteins

  15. Phase Switch Promoter System -> for very toxic proteins -> inversion induced during infection FimA interacts with receptor on macrophages -> blocks ingestion -> inversion carried out by FimB and FimE

  16. Phase Switch Promoter System -> for very toxic proteins -> expression of Pap (Pili) controlled by transcriptional active and inactive states -> Off state: repression of papBA promoter by Lrp and H-NS -> differencies in methylation pattern of 2 sites -> switch on/off

  17. Phase Switch Promoter System -> for very toxic proteins -> expression of 2 different sets of proteins -> determine phage host range -> Gin invertase responsible for switch

  18. Bacillus Flagellar stains of various species of Bacillus from CDC In 1872, Ferdinand Cohn, a student of Robert Koch, recognized and named the bacterium Bacillus subtilis. The organism was made to represent a large and diverse genus of Bacteria, Bacillus,  and was placed in the family Bacillaceae. The family's distinguishing feature is the production of endospores, which are highly refractile resting structures formed within the bacterial cells. Since this time, members of the genus Bacillus are characterized as Gram-positive, rod-shaped, aerobic or facultative, endospore-forming bacteria.

  19. Bacillus • Antibiotic Producers: B. brevis (e.g. gramicidin, tyrothricin), B. cereus (e.g. cerexin, zwittermicin), B. circulans (e.g. circulin), B. laterosporus (e.g. laterosporin), B. licheniformis (e.g. bacitracin), B. polymyxa (e.g. polymyxin, colistin), B. pumilus (e.g. pumulin) B. subtilis (e.g. polymyxin, difficidin, subtilin, mycobacillin). • Pathogens of Insects: B. larvae, B. lentimorbis, and B. popilliae are invasive pathogens. B. thuringiensis forms a parasporal crystal that is toxic to beetles. • Pathogens of Animals: B. anthracis, and B. cereus.  B. alvei, B. megaterium, B. coagulans, B. laterosporus, B. subtilis, B. sphaericus, B. circulans, B. brevis, B. licheniformis, B. macerans, B. pumilus, and B. thuringiensis have been isolated from human infections. • The Genus Bacillus includes two bacteria of significant medical importance, B. anthracis, the causative agent of anthrax, and B. cereus, which causes food poisoning. Nonanthrax Bacillus species can also cause a wide variety of other infections, and they are being recognized with increasing frequency as pathogens in humans.

  20. Bacillus Endospores Bacillus thuringiensis phase micrograph Bacillus anthracis Crystal violet stain viewed by light microscopy Spore stain of a Bacillus species. CDC. Mature spores stain green, whether free or still in the vegetative sporangium; vegetative cells and sporangia stain red.

  21. Bacillus • Bacillus strains used as production organisms: - B. subtilis - B. brevis - B. licheniformis • Transformation systems: - via competent cells (during transition from vegetative cells -> sporulation, cell can take up DNA (ss) when population reaches a metabolic state called competence) - protoplast - plasmid rescue - bacteriophage-mediated transduction • Vectors: - replicating plasmids (pUB110, pE194, pC194, pHP13, shuttle vectors) -> replicating plasmids with temperature-sensitive origin of replication (replication stops above certain temp. -> pE194 stops above 45ºC) - integrative vectors (normally shuttle vectors)

  22. Transformation Systems

  23. Replicating Plasmids:

  24. Replicative Shuttle Vectors:

  25. Integrative Plasmid No replicon for Bacillus -> selection in Bacillus driggers integration -> useful if construct should carry one copy of foreign gene -> integration of circular plasmid -> shuttle vectors

  26. Integrative Plasmid No replicon for Bacillus -> selection in Bacillus driggers integration -> useful if construct should carry one copy of foreign gene -> integration of linear plasmid -> generate deletions -> shuttle vectors

  27. Bacillus • Promoters: - aprE promoter -> induction with onset of sporulation - amylase promoter -> growth-phase and nutrition regulated promoter (induction at end of exponential growth + repression by glucose) - sacB promoter (levansurase) -> not regulated - spac promoter -> hybrid promoter (subtilis phage + lac operator) -> induction with IPTG - T7 system -> hybrid system -> E. coli T7 system (IPTG induction) + T7 polymerase (on chromosome) under control of a xylose- inducible promoter - xylose-inducible promoter -> induction with xylose, strong promoter (200 times), repressed by glucose -> Bacillus is good secreter -> Signal sequence longer and N-terminus more pos. charged -> low GC-content bacteria

  28. Bacillus as expression host

  29. Bacillus as expression host

  30. Other Bacteria for Gene expression: Gram positive: Staphylococcus, Streptococcus, Lactobacillus, Streptomyces,... Gram negative: Pseudomonas, Agrobacterium, Xanthomonas,...

  31. Transposable Gene expression systems: E. coli carrying transposon -> through conjugation into other Gram-negative bacteria

  32. Products produced in Prokaryotic Systems • Restriction Endonucleases -> produced in E. coli • L- Ascorbic Acid (Vitamin C) -> recombinant Erwinia herbicola (gram-negative bacterium) • Synthesis of Indigo (blue pigment -> dye cotton /jeans) -> produced in E. coli • Amino Acids-> produced in Corynebacterium glutamicum (gram-positive bacterium) • Lipases (laundry industry) -> from Pseudomonas alcaligenes produced in Pseudomonas alcaligenes • Antibiotica (most of them from Streptomyces, other gram-positive bacteria, fungi) -> produced in recombinant Streptomyces and fungi (Penicillium) • Biopolymers (PHB -> biodegradable plastics) -> produced in E. coli (stabilized with parB)

More Related