TightRegulation, Modulation, and High-Level Expression byvectors ContainingtheArabinose Promoter

Transcription

1 TightRegulation, Modulation, and High-Level Expression byvectors ContainingtheArabinose Promoter L M Guzman et al. (1995) Journal of Bacteriology 177:

2 Outline 1. Introduction 2. Objective 3. Results 4. Summary 5. Take-Home-Lesson 6. Discussion and current questions

3 Arabinose operon Araoperon is from E. coli Activated by the presence of arabinose Repression in presence of glucose The regulator protein is AraC promoter regulate the expression

4 Activationofthe promoter AraC forms a complex with arabinose and bindsataraiasa dimer ara arai Watson, JD, et al.: Molecular Biology of the Gene, Ed. 6, San Francisco, 2008, Pearson Education, p. 567

5 Activation ofthe promoter CataboliteActivatorProtein (CAP) formsa complexwithcampandbindson the CAP site CAP avoidstheformationofa DNA loop CAP site Watson, JD, et al.: Molecular Biology of the Gene, Ed. 6, San Francisco, 2008, Pearson Education, p. 567

6 Repression ofthe promoter AraCwithoutbindingtoarabinoseadoptsa different conformation Binding toara andara leadstothe formationofa DNA loop ara arai Watson, JD, et al.: Molecular Biology of the Gene, Ed. 6, San Francisco, 2008, Pearson Education, p. 567

7 Vectors aracgeneand promoter from arabinose operon MCS: multiple cloning site Ori: origin of replication Resistance gene is for instance Ampicillin

8 Outline 1. Introduction 2. Objective 3. Results 4. Summary 5. Take-Home-Lesson 6. Discussion and current questions

9 Objective Assessment oftheabilityof vectorsfor regulation, modulation and overexpression of recombinantproteinsin E. coli.

10 Outline 1. Introduction 2. Objective 3. Results 4. Summary 5. Take-Home-Lesson 6. Discussion and current questions

11 1) Inductionandrepressionofthe vector The phoageneis cloned into the vector phoa encodes for alkaline phosphatase (AP) Quantity of the produced AP monitors the expression level

12 Repression Comparison of two cultures of strains: KS272 ara wild-type LMG194 ara cannot metabolize arabinose Grownon minimal medium + glycerol+ arabinose, which is shifted to glucose

13 HowtodetecttheproducedAP: Pulse-labelling: samples are incubated with - methionine, washed, centrifuged and resuspended in medium with unlabelled methionine Immunoprecipitation: precipitation of the protein with a specified antibody Gel electrophoresis Fluorography: Take a X-ray image of the gel

14 Fluorography of the gel Resultant repression AP OmpA AP OmpA Strain Repression Reason ara fast Arabinoseisdegraded ara slow Arabinose concentration still the same

15 Induction Experiment: Cultures grow on glucose containing medium, which is shifted to arabinose Result: Both strains are induced fast.

16 Conclusions The repressionofthe promoterworksfast and efficient. The induction rate of the promoterisveryfast.

17 2) The ratio of induction / repression Comparison of different vectors in RM and MM: Plasmid Attributes Gene Strains plmg163 - plmg162 Additional SDbox, 5 residues from arab pdb3 SD-boxis optimized plmg281 -promoter ftsq-phoa fusion ftsq-phoa fusion ara, ara ara, ara phoa ara, ara ftsq-phoa lac fusion

18 The ftsq gene FtsQgene encodes a cell division membrane protein It is generally expressed at low levels ftsq-phoa fusion: Original SD-box from ftsqfor translation Recombinant protein works like normal AP

19 Observations: Comparison results Vectors with an optimized or an additional SD-box reach a higher expression level The vectors have ratios between 118 and 1855; Repression works more efficient The vector has a ratio of 50

20 Conclusions Some vectors are qualified for high-level expression The repression of the promoter is highly effective

21 3) Modulation of the vectors Why is modulation important?

22 Modulation of the vectors Modulation is very useful to receive several expression levels Experiment: Quantifying the expression by different concentrations of arabinose Observation: Between inducer concentration and expression level exists a partial linear correlation

23 Results and conclusions ara MM ara RM Modulation of the strains occurs elsewise in minimal medium Mutants have a more effective expression in minimal medium Their modulation is highly efficient and reproducible

24 4) vectors to examine null mutations Study of null mutants of essential genes is difficult The mutant keeps a complementing vector, which must be turned off very effectively Complementation get to occur partly -> null phenotype The induction with arabinose determines the viability of the mutants

25 Null mutation experiment Chromosomal null mutation of the ftsl gene ftslgene: encodes for a cell division and growth membrane protein Plasmids contain the ftsl gene Additional mutation of the pcnb gene

26 The pcnb gene pcnb: Poly(A) polymerase, controls the plasmid copy number (pcn) An anti-sense strand blocks the primer -> no replication Polyadenylationof the anti-sense strand is a marker for degradation -> replication can start pcnbmutants determine low-copy-plasmid and plasmidless cells

27 Growth of both strains in presence of arabinose pcnbmutant do not grow in glucosecontaining medium no complementation of ftsl gene Fundamentality of the ftsl gene

28 Conclusions Study of phenotypes from null mutations by reducing the gene dosage of ftsl Reducing occurs by low expression levels and repression of the promoter

29 Outline 1. Introduction 2. Objective 3. Results 4. Summary 5. Take-Home-Lesson 6. Discussion and current questions

30 Summary 1) The arac- systems are repressed rapid and efficient; their induction rate is very fast 2) Some vectors qualified for high-level expression 3) Modulation is very effective and reproducible, especially for mutants in minimal medium 4) Study of null phenotypes by a strict regulation of the vector

31 Outline 1. Introduction 2. Objective 3. Results 4. Summary 5. Take-Home-Lesson 6. Discussion and current questions

32 Take-Home-Lesson The promoter from arabinose operon is used for strict regulation, modulation and overexpression of recombinant proteins.

33 Outline 1. Introduction 2. Objective 3. Results 4. Summary 5. Take-Home-Lesson 6. Discussion and current questions

34 Discussion and current questions 1) Was versteht man unter einem depletion assay? 2) Warum liegen high copy number Plasmide vom ColE1-Typ in einer E. coli pcnb-mutante in geringer Kopienzahl vor?

35 References L M Guzman, D Belin, M J Carson, and J Beckwith Tight Regulation, Modulation, and High-Level Expression by Vectors Containing the Arabinose Promoter. J. Bacteriol. 177: M. J. Carson, J. J. Barondess, and J. Beckwith The FtsQ protein of Escherichia coli: membrane topology, abundance, and cell division phenotapesdue to overproduction and insertion mutations. J. Bacteriol. 173: Watson, JD, et al.: MolecularBiologyoftheGene, Ed. 6, San Francisco, 2008, Pearson Education, p W. S. Klug, et al.: Genetik, Ed. 8, München, 2007, Pearson Studium, p ( ) ( )