unit ii lecture 4 b. tech. (biotechnology) iii year v th semester ebt-501, genetic engineering
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Unit II Lecture 4
B. Tech. (Biotechnology) III Year V th Semester
EBT-501, Genetic Engineering
Unit II
• Restriction modification• Enzymes used in recombinant DNA technology
endonucleases, ligases and other enzymes useful in gene cloning
• PCR technology for gene/DNA detection, cDNA,• Use of Agrobacterium for genetic engineering in
plants• Gene libraries; Use of marker genes. • Cloning of foreign genes: DNA delivery methods -
physical methods and biological methods, • Genetic transformation of prokaryotes: Transferring DNA
into E. coli –Chemical induction and Electroporation,
Gene Transfer in Plants
The Ti plasmid of Agrobacterium tumefaciens is an important tool for
transferring genes into plants.
Agro bacterium tumefacians is a bacterium that causes a disease known as crown gall in plants.Infects plants by transferring its genetic material into plant cell.Agrobacterium transformation is the most common technique for genetically engineered plants
Agro-bacterium tumefacians
Production of Transgenic Plants
• Microprojectile bombardment• Electroporation• Agrobacterium tumefaciens-mediated
transformation
• Plant cells are totipotent, so an entire plant can be regenerated from a genetically engineered plant somatic cell.
Agrobacterium tumifaciens Infection
The Ti Plasmid
Transformation of Plant Cells by the Ti Plasmid
Key steps from natural Agrobacterium to “useful”
Agrobacterium• Some vir genes deleted--disarmed
– Opines not going to be produced– Deleting tumorogenesis function
• Choosing strains that transfer DNA in lab
• Clone in genes of interest, antibiotic resistance genes, etc.
• Binary system-- two plasmids are better than one Ti plasmid
Plant transformation with the Ti plasmid of Agrobacterium tumefaciens
• Tumor formation is the result of the transfer, integration and expression of genes on a specific segment of A. tumefaciens plasmid DNA called the T-DNA (transferred DNA)
• The T-DNA resides on a large plasmid called the Ti (tumor inducing) plasmid found in A. tumefaciens
The Ti plasmid of Agrobacterium tumafaciens and the transfer of its T-DNA to the plant nuclear
genome
Ti plasmid: structure & function• The infection process:• Wounded plant cell releases
phenolics and nutrients.• Phenolics and nutrients cause
chemotaxic response of A. tumefaciens
• Attachment of the bacteria to the plant cell.
• Certain phenolics (e.g., acetosyringone, hydroxyacetosyringone) induce vir gene transcription and allow for T-DNA transfer and integration into plant chromosomal DNA.
• Transcription and translation of the T-DNA in the plant cell to produce opines (food) and tumors (housing) for the bacteria.
• The opine permease/catabolism genes on the Ti plasmid allow A. tumefaciens to use opines as a C, H, O, and N source.
The binary Ti plasmid system involves using a small T-DNA plasmid (shown below) and a disarmed (i.e., no T-DNA) Ti plasmid in A.
tumefaciens
DNA-delivery methods
Method Comment
Ti plasmid-mediated gene transfer *
Excellent and highly effective, but limited to dicots
Microprojectile bombardment * Easy and effective; used with a wide range of plants
Viral vectors Not very effective
Direct gene transfer into plant protoplasts
Only certain protoplasts can be regenerated into whole plants
Microinjetion Tedious and slow
Electroporation * Limited to protoplasts that can be regenerated into whole plants
Liposome fusion Limited to protoplasts that can be regenerated into whole plants
Microprojectile bombardment or biolistic-mediated DNA transfection equipment
• equipment(a) lab version(b) portable version
• When the helium pressure builds to a certain point, the plastic rupture disk bursts, and the released gas accelerates the flying disk with the DNA-coated gold particles on its
• lower side. The gold particles pass the stopping screen, which holds back the flying disk, and penetrate the cells of the plant.
Some plant cell reporter and selectable marker gene systems
Enzyme activity Selectable marker
Reporter gene
Neomycin phosphotransferase (kanr) Yes Yes
Hygromycin phosphotransferase (hygr) Yes Yes
Nopaline synthase No Yes
Octopine synthase No Yes
-glucuronidase (GUS) No Yes
Firefly luciferase No Yes
-galactosidase No Yes
Bromoxynil nitrilase Yes No
Green fluorescent protein (GFP) No Yes
Genetically Modified Organisms
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