B. Tech. (Biotechnology) III Year V th Semester 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 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

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

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 Hygromycin phosphotransferase (hygr) Nopaline synthase No Octopine synthase -glucuronidase (GUS) Firefly luciferase -galactosidase Bromoxynil nitrilase Green fluorescent protein (GFP)

Genetically Modified Organisms