TRANSFER MATERI GENETIKSri Agung Fitri Kusuma

MEKANISME TRANSFER MATERI GENETIK

Bacterial transformationTransformation is the act of putting foreign DNA into a bacterial cellOccurs in nature, but VERY RARELY!Weve perfected an artificial method to force bacteria to take up DNA

TRANSFORMASIProses perpindahan DNA bebas dari lingkungan ke sel resipien yang kompetenCompetence: alterations in the bacterial cell wall that make it permeable to large DNA molecules Sel kompeten mengikat DNA bebas 1000 kali lebih tinggi dibandingkan non kompetenNaturally transformable : dapat mengambil DNA bebas dari lingkungannya tanpa perlakuan khusus -Natural competence : Acinetobacter, azotobcate, bacillus, Streptococcus, Haemophilus, Neisseria dan Thermus.Transformasi yang perlu perlakuan khusus supaya mudah tertransformasi, pada E. coli dan beberapa bakteri Gram (-); lingkungan dengan konsentrasi kalsium yang tinggi dan didinginkan beberpa menit, atu menggunakan electroporation.Sel kompeten mengikat DNA bebas 1000 kali lebih tinggi dibandingkan non kompeten.Efisiensi DNA uptakeSpesifity of DNA uptake, H. influenzae (5 AAGTGCGGTCA 3), N. gonorrhoeae (5 GCCGTCTCAA 3), only from their own species and others can take up DNA from any sources.DNA uptake during growth cycle, but most naturally transformable bacteria usually just before they reach stationary phase

Transformation

GRIFFITH'S EXPERIMENT

Cells in mid-logarithmic phase are suspended in cold calcium chloride which makes them permeable to DNAThey are mixed with the DNA to be insertedThey are heat shocked which drives the DNA into the cellThey are allowed to recover at 37oCThey are spread on a plate containing a selection drug(s)Transformation procedureprobable mechanism

Transformation procedureE. coliCaCl2cold!+42oCRecover at 37oCIncubate at 37oC overnightLB Amp+KanLB Amp+Kan

Transformation procedureE. coliCaCl2cold!+42oCRecover at 37oCIncubate at 37oC overnightLB Amp+KanLB Amp+KanMaking cells competent

Heat shock

elektroforasi

Transformation

Important considerationsCells must be in log phase when making competent!Cells must be kept cold!Cells must recover after heat shock!

Electroporation

Gene gun methodDNATransformed plant cellSource: Monsantoplant chromosomeinserted genepellets

ConjugationDefinition: Gene transfer from a donor to a recipient by direct physical contact between cellsMating types in bacteriaDonorF factor (Fertility factor)F (sex) pilusRecipientLacks an F factor

PlasmidsDefinition: Extrachromosomal genetic elements that are capable of autonomous replication (replicon)Episome - a plasmid that can integrate into the chromosome

Classification of PlasmidsTransfer propertiesConjugativeNonconjugativePhenotypic effectsFertilityBacteriocinogenic plasmidResistance plasmid (R factors)

Conjugation and F+ factorF+ factor (fertility factor)- a sophisticated plasmid OriT, Origin of transfer insertion sequences, genes for own replication, genes for own transfer

Mechanism of F x F- CrossesDNA transferOrigin of transferRolling circle replication Pair formation Conjugation bridge

Conjugation converts an F- to F+, and original F+ remains F+Note: no host genes (in blue) transferred from F+ to F- Conjugation cycle

Hayes expt:Take strain A, which is streptomycin-resistant, and auxotrophic for biotin and methionine.Take strain B, which is streptomycin-sensitive, and auxotrophic for threonine and leucine.Mix the two on a minimal-medium plate containing streptomycin.Wait and see.

MCB 140 09-19-07 *15.11J. Lederberg, E. Tatum (1946) Novel genotypes in mixed cultures of biochemical mutants of bacteria. Cold Spring Harbor Symp. Quant. Biol. 11: 113-114.

MCB 140 09-19-07 *Interruption de la pntration de l'ADN mle dans le cytoplasme de la bactrie femelle l'aide d'un warring blender (simple mixer mnager).Cours de Physiologie et de gntique bactriennes, 1957 Illustration humoristique des cours par une srie de dessins de F. Lavall Lgendes de Georges Cohen http://www.pasteur.fr/infosci/archives/mon/im_laval.html

TRANSDUKSIProses transfer DNA asing (bukan berasal dari DNA virus itu sendiri) ke dalam bakteri melalui bakteriofaga yang mengandung DNA asing tersebutGeneralized and specialized transductionGeneralized transduction, setiap segmen dari DNA bakteri donor dapat ditransferSpecialized transduction, hanya gen tertentu yang dekat dengan situs perlekatan lysoghenic phage saja yang dapat ditransfer.Transducing phage, phages capable of transductionDonor and recipient strainTransductant, cells that have received DNA from another bacterium by transduction

TransductionTypes of transductionGeneralized - Transduction in which potentially any dornor bacterial gene can be transferred. Specialized - Transduction in which only certain donor genes can be transferred

Generalized TransductionRelease of phage Phage replication and degradation of host DNAAssembly of phages particlesInfection of recipientLegitimate recombination Infection of Donor

A second form of gene transfer- transduction

SPESIALIZED TRANSDUCTION

Three mechanisms of gene transfer in bacteria

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