RECOMBINANT DNA TECHNOLOGY

Genetic manupulation” 1. Cellular manupulation involving culture of cells(haploid cells) andhybridization of somatic cells (protoplast fusion) 2. Molecular manupulation involving construction of rDNA molecules and their insertion in to a vector and their establishment in to a host cell or organism.

What is recombinant DNA technology? The procedure is to cut the DNA of the donor organism into pieces with restriction enzymes, and insert one of these fragments into the DNA of the host. Most of the time a bacterial or virus plasmid is used to insert the donor DNA. A plasmid is a circular DNA fragment, which can be opened with the same restriction enzymes as the DNA fragment of the donor. A plasmid containing DNA from the donor is called a vector. The recombinant vector can then be used to transform bacterial or virus cells. These bacterial cells are plated and colonies are grown. What is recombinant DNA? Recombinant DNA is the formation of a novel DNA sequence by the formation of two DNA strands. These are taken from two different organisms. These recombinant DNA molecules can be made with recombinant DNA technology.

I. Gene cloning The enzyme DNA ploymerase functions at sites known as “origin of replication” which is usually but absent in small fragment of DNA thus vectors are used. E.g plasmid. Restriction enzymes works at Recognition site Both foreign DNA and vectors are cut by same enzyme

II. Selection of recombinant bacteria 1. Complementation of genetic defects in nutrition 2. Selection of a suitable character 3. By nucleic acid hybridization technique

III. Transcription of foreign gene

IV. Translation of foreign gene Splicing mRNA

V. Post transcriptional modification

Expression in different hosts

Plasmids and Vectors DNA or RNA Single /double stranded Circular or linear 1.5 to 1500 kb Plasmids are self replicating double stranded extrachromosomal DNAs maintained as independent molecules by most bacteria

Classification of plasmid 1. Conjugative or transmissible plasmid: plasmid can mediate their own transfer from one bacteria to another( contains tra and mob regions), found in gram –ve and some gram +ve bacteria 2. Non-conjugative or non-transmissible plasmids: nbut can be mobilized by conjugative plasmid of the same cell.

Downstream Processing Recovery and purification of biosynthetic products. Analytical bioseparation

Steps in downstream processing 1. Removal of insolubles: filtration, sedimentation, precipitatiuon. Grinding, leaching, homogenization. 2. Product Isolation: Removal of those components whose property vary markedly than that of the desired product. E.g water. Solvent extraction, adsorption, ultrafiltration etc. 3. Product purification: Removal of thos components that resembles the product closely. Expensive, sensetive sophisticated. Affinity, size exclusion, reverse phase chromatography, crystallization etc. 4. Product polishing: final processing steps. Stable, easily tranportable and convenient. Lyophilization, sterilization. Quality control

Types of bioreactors: Stirred tank Airlift Combinations and variations of both Membrane

Scale up considerations: oxygen masstransfer, power consumption, shear rate, impeller speed Enhance capacity and speed

Platform Technologies Rapid and economic process development and scale up Rhobust process and better technology transfer Reduca validatin cost PCR, Anion exchange steps

a.Resin improvements b.Increased velocity c.Increased capacity d.Overall productivity e.Selectivity and purity f.Cleaning and sanitization