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Escherichia coli General Information Escherichia coli is a rod-shaped bacterium. Each bacterium measures approximately 0.5 μm in width by 2 μm in length.

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Presentatie over: "Escherichia coli General Information Escherichia coli is a rod-shaped bacterium. Each bacterium measures approximately 0.5 μm in width by 2 μm in length."— Transcript van de presentatie:

1 Escherichia coli General Information Escherichia coli is a rod-shaped bacterium. Each bacterium measures approximately 0.5 μm in width by 2 μm in length. E. coli is a Gram-negative bacterium. E. coli cells stain Gram negative because they have a thin cell wall with only 1 to 2 layers of peptidoglycan. E. coli is a facultative anaerobe, which means it does not require oxygen, but grows better in the presence of oxygen. Much like "breeds" of dogs, there are "strains" of Escherichia coli. Many strains of E. coli are harmless and are important to good digestive health since they reside in the large intestine and provide their hosts with vitamin K. Other strains, which are referred to as pathogenic strains, are harmful and produce toxins. Taxonomy E. coli is a member of the: Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Escherichia.

2 Escherichia coli Major subgroups E. coli strains fall into five major taxonomic lineages (A, B1, B2, D, and E; ). In addition, species in the genus Shigella group within the E. coli groupings and different Shigella species have different E. coli groups as their closest relatives. E. coli K-12 strains are in subgroup A. Types E. coli types are based on major surface antigens O-antigen: the O antigen is part of the lipopolysaccharide. O antigens were originally defined by reaction with antisera from rabbits immunized with boiled bacteria. Hundreds of O groups have been identified. H-antigen: H antigen is flagellin. K-antigens: K antigens were named for capsule (Kapsel in German). Sera subdivide K antigens into L, B, and A groups. At the molecular level, there are two major kinds of K antigens - polysaccharide K antigens - protein K antigens, mostly fimbriae R-antigen

3 Escherichia coli StrainsO antigenH antigenK antigenNotes C B K-12O16K-12 is phenotypically rough. (mutant)Note that K-12 does NOT refer to an antigenic type, and should therefore be hyphenated. Complementation using WG1 shows that the parent of laboratory K-12 strains was of type O16.

4 MG , volledige genoomsequentie van E.coli K12 (Blattner et al.) MC1061startpunt van Doug Hanahan voor de constructie van DH1 en verder tot DH10B belangrijke elementen in deze stappen : recA1 : verbeteren van de kloonstabiliteit (homologe recombinatie verhinderen) endA1 : inactiveert de codering van het periplasmatisch DNA-specifieke endonuclease => verhoogt de DNA stabiliteit tijdens transformatie een defectief  80 faag met lacZ  M15 voor screening door  -complementatie een deoR mutatie zou aanwezig zijn die verhoogde transformatie-efficiëntie geeft, maar dit blijkt nu op een foute interpretatie te berusten. (DeoR = repressor van het deo operon (betrokken bij het nucleoside catabolisme) (er blijken nu 52 kandidaat-gemuteerde-genen te zijn potentieel verantwoordelijk voor dit interessante fenotype) DH10 DH10B : na Tn10 insertie (in mdoB) werd met 2-carboxyl, 5-butyl pyridine (5-butyl picoline of ‘fusaric acid’ : heeft antivirale eigenschappen) de Tc R (van het Tn10) tegengeselecteerd (i.a.v. een plasmide dat recA tot expressie bracht, en nadien verwijderd werd). Met het verwijderen van de Tn10 merker werd in het isolaat DH10B het flankerende DNA met de MDRS loci gedeleteerd (mrr, mcrA, mcrB, mcrC). Dit verhoogt de efficientie van klonering van zoogdier DNA, waarin cytosine vaak gemethyleerd is. D10B tonA : spontane mutant die werd geselecteerd. tonA geeft resistentie tegen de fagen T1, T5 en  80. Is commercieel verkrijgbaar (Invitrogen NV)

5 Strain Information E. coli K-12 MG1655 Description Genotype: F - lambda - ilvG rfb-50 rph-1 Serotype: OR:H48:K - This strain was sequenced by the Blattner laboratory because it approximates wild-type E. coli and "has been maintained as a laboratory strain with minimal genetic manipulation, having only been cured of the temperate bacteriophage lambda and F plasmid by means of ultraviolet light and acridine orange, respectively." (Blattner, et al. 1997). The mutations listed in the genotype are present in most K-12 strains and were probably acquired early in the history of the laboratory strain. - A frameshift at the end of rph results in decreased pyrE expression and a mild pyrimidine starvation, such that the strain grows 10 to 15% more slowly in pyrimidine-free medium than in medium containing uracil. -The ilvG mutation is a frameshift that knocks out acetohydroxy acid synthase II. -The rfb-50 mutation is an IS5 insertion that results in the absence of O-antigen synthesis. MG1655 was derived and named by Mark Guyer from strain W1485, which was derived in Joshua Lederberg's lab from a stab-culture descendant of the original K-12 isolate. This original E. coli strain K-12 was obtained from a stool sample of a diphtheria patient in Palo Alto, CA in 1922.

6 De co-lineariteit tussen MG1655 en DH10B is behoorlijk, maar wat vooral opvalt is de veel hogere mutatiesnelheid in DH10B. De mutatiesnelheid van DH10B is 13,5 maal hoger t.o.v. MG1655. Dit blijkt echter niet te wijten aan puntmutaties, noch aan deleties, maar aan IS-elementen, voornamelijk IS150. Er zijn 226 gemuteerde genen in DH10B tegenover MG1655, vooral als gevolg van de genetische manipulaties (transducties). Er is enige “remodelling” van de genoomarchitectuur, o.a. een bp tandem herhaling. DH10B is een Leu-auxotroof (deletie van leuLABCD) : groeit op minimaal medium alleen i.a.v. leucine. De groeisnelheid is lichtjes trager dan van de wild-type stam.

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8 Meestal transductie met faag P1 vanuit E.coli K12 stammen, behalve met D7091F die een E.coli B stam is.

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