Replikation af Vibrio cholerae: Replication of Vibrio cholerae chromosome II

Hoda Al-Ansari, Ala Al-Najar, Simon Lynge Hansen, Andreas Skovgård Jacobsen, Maiken Nielsen & Andreas Thymann

Studenteropgave: Semesterprojekt

Abstrakt

Abstrakt: Bakterien Vibrio choleraes genom er fordelt på to kromosomer, et stort og et mindre, som navngives henholdsvis. kromosom I og kromosom II. Kromosom I origin, oriCIVc, ligner det origin, der findes i Escherichia coli, oriC, hvorimod origin på kromosom II, oriCIIVc, både har kromosom- og plasmidlignende egenskaber. Dette tyder på, at kromosom I er det originale kromosom og kromosom II er et megaplasmid, som senere er blevet integreret i cellen som et kromosom. Projektet omhandler, hvorvidt et minikromosom med oriCIIVc replikeres som et plasmid eller et kromosom. Dette undersøges ved at sætte en kultur af E. coli med minikromosomet under selektivt pres fra forskellige koncentrationer af kanamycin. Plasmider har en meget streng kopitalskontrol, og det selektive pres vil ikke øge kopitallet. Derimod har et minikromosom ikke nogen kopitalkontrol, og vil replikeres hyppigere, desto større det selektive pres er. Da kopitallet af oriCIIVc nedreguleres af et inkompatibelt område, inc, ønskes dette fjernet for at øge kopitallet. Da dette mislykkedes, benyttede vi et allerede konstrueret oriCIIVc minikromosom med en copy-up mutant i initiator proteinet, RctB. Ved lave kanamycinkoncentrationer viste det sig, at minikromosomet hyppigere blev replikeret end ved høje koncentrationer. Det kunne derfor ikke påvises, om kromosom II replikeres som et plasmid eller et kromosom. Ud fra litteraturen antydes det, at kromosom II primært replikeres som et plasmid, dog er denne replikation timet til replikationen af kromosom I. Abstract: The genome of Vibrio choleraes is distributed on two chromosomes, one large and one small, chromosome I and chromosome II. The origin of chromosome I, oriCIVc, reflects the same origin as on E. coli, oriC, whereas the origin of chromosome II, oriCIIVc, has both plasmid and chromosome like abilities. This indicates that chromosome I is the original chromosome and chromosome II was a mega plasmid, that later was integrated in the cell as a chromosome. This project deals with whether a minichromosome with oriCIIvc replicates as plasmid or as a chromosome. This is examined by putting a culture of E. coli, containing the minichromosome, under selective pressure from different concentrations of kanamycin. Plasmids have a very strict copy number control, and selective pressure will not increase the copy number. A minichromosome has no copy control, and it will replicate far more the greater the selective pressure becomes. Because the copy number of oriCIIvc is down regulated by an incompatible area, inc, it is wanted that this area is to be removed to increase the copy-number. The attempt failed and instead we used an already constructed orIICIvc minichromosome, with a copy-up mutation in the initiator protein, RctB. It was here shown that the minichromosome replicated more at lower kanamycin concentrations, than at high concentrations. Because of this, we could not prove if the plasmid replicated as a plasmid or a chromosome, because the results showed that the replication lowered as a function of the kanamycin concentration. From the litterateur it can be concluded that chromosome II replicates as a plasmid, although it is timed to the replication of chromosome I.

UddannelserMolekylærbiologi, (Bachelor/kandidatuddannelse) Bachelor el. kandidat
SprogDansk
Udgivelsesdato20 jan. 2010

Emneord

  • Vibrio cholerae
  • oriCII