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