LU3 Microbiology Sem 2 2022 PDF

STB1073 STU 1013 LU3 MODERN BIOTECHNOLOGY: MULTIDISCIPLINARY INPUT BIOCHEMISTRY GENETICS MICROBIOLOGY CELL BIOLOGY  General strategy in Biotechnology (using STB1073...

STB1073 STU 1013 LU3 MODERN BIOTECHNOLOGY: MULTIDISCIPLINARY INPUT BIOCHEMISTRY GENETICS MICROBIOLOGY CELL BIOLOGY  General strategy in Biotechnology (using STB1073 Microbiology) 5’ 3’ Clone gene Insert into suitable vector Transform host cells Select transformed cell Culture transformed clone Extract and purify recombinant product  STB1073 What is a Cloning vector ? A cloning vector is a DNA molecule that carries a foreign DNA into a host cell (usually bacterial or yeast) Character – able to replicate, producing many copies of itself along with the foreign DNA.  STB1073 Types of Cloning Vectors A few examples: Plasmid Phage Cosmid Bacterial Artificial Chromosomes (BAC) Yeast Artificial Chromosomes (YAC)  STB1073 Plasmids Plasmids are small, circular, extrachromosomal DNA molecules found in bacteria, which can replicate on their own, outside of a host cell. They have a cloning limit of 100 to 10,000 base pairs or 0.1-10 kilobases (kb). A plasmid vector is made from natural plasmids by removing unnecessary segments and adding essential sequences.  STB1073 Plasmids Phenotypes that can confer advantage to a host cell include  Antibiotic resistance, Antibiotic production  Sugar fermentation for energy  Degradation of aromatic compounds for energy  Heavy metal resistance  Toxin production  STB1073 A typical E. coli plasmid vector STB1073  STB1073 Phages Derivatives of bacteriophage lambda (λ phage), a virus which infects E. coli. Linear DNA molecules, which its region can be replaced with foreign DNA without disrupting its life cycle. Major advantage of the λ phage vector is its high transformation efficiency, which is about 1000 times greater than that of the plasmid vector. They also have a larger cloning limit than plasmids, consisting of 8-25 kb. STB1073 STB1073  STB1073 Cosmids Cosmids are extrachromosomal circular DNA molecules that combine features of plasmids and phages. They also have a high transformation efficiency. The cloning limit of 35-50 kb is larger than that of plasmids or phages STB1073  STB1073 Bacterial Artificial Chromosomes (BAC) BACs are based on bacterial plasmids, which are small pieces of bacterial DNA that give the bacteria the ability to initiate conjugation with adjacent bacteria. They have a cloning limit of 75-300 kb.  STB1073 Examples of BAC:  STB1073 Yeast Artificial Chromosomes (YAC) YACs are artificial chromosomes that replicate in yeast cells. They are hybrids of bacterial plasmid DNA and yeast DNA. YACs are grown in the yeast Saccharomyces cerevesiae and so contain selectable markers which are suitable for the host system. They are the largest of the cloning vectors, with a cloning limit of 100-1000 kb, however they have very low efficiency. STB1073  STB1073 Microorganisms as expression hosts Hosts for production of recombinant products proteins, enzymes products of multi-enzyme pathways  Host requirements Suitable vectors Effective transformation Ready fermentation and scale-up capacity  STB1073 Microbial expression hosts Escherichia coli (E. coli) Saccharomyces cerevisiae Bacillus spp  STB1073 E. coli ADVANTAGES DISADVANTAGES Numerous specific vectors Low expression yields (l phage and pUC plasmid derivatives)  Very high transformation efficiencies.  Very well understood fermentations Simple cell recovery and lysis  STB1073 Saccharomyces cerevisiae ADVANTAGES DISADVANTAGES Moderate expression Low-medium yields transformation Very well understood efficiency fermentations Limited range of Simple cell recovery and vector systems lysis rGenes must be stably incorporated  STB1073 Bacillus spp. ADVANTAGES DISADVANTAGES High transformation Limited range of efficiencies vector systems Very high expression Optimised vector yields (>10 g/L) systems are Simple fermentations proprietary Simple cell recovery Bacillus subtilis  STB1073 Expression development Multicopy plasmids Multiple selective (resistance) markers (Ampicillin, Tetracycline, Kanamycin, Thiostreptin) Different promoters (Trp, temperature-inducible) Multiple promoters (shuttle vectors for different hosts) Multiple ori sequences N-terminal tags (e.g., polyHis) STB1073  STB1073 Viral Vectors A viral vector is a virus that carries a modified or foreign gene. They are commonly used in gene therapy where the viral vector delivers the desired gene to a target cell. Some of the viruses used as vectors in gene therapy include: Retroviruses Adenoviruses Parvoviruses Herpesviruses Poxviruses  Contribution of Microbiology to STB1073 Biotechnology Supply of antibiotics Cloning Process – as Vectors (Carrier of stretches of nucleic acids) and as hosts Industrial enzymes – eg: amylase, cellulase Industrial products – biodiesel, acetone, acetic acid Food and beverages industries. Research enzymes – for molecular work Medicine – Vaccine production, gene therapy Agriculture  STB1073 Agrobacterium in Biotechnology Bacteria that causes tumours in plants. The ability of Agrobacterium to transfer genes to plants has been exploited for genetic engineering for plant improvement. A large (200 kb) circular DNA plasmid is responsible for the production of the tumour and the transforming abilities. This tumour inducing (Ti) plasmid transfers and inserts a region of the plasmid, called T-DNA, into the host chromosome. The plasmid is 'disarmed' by deletion of the tumour inducing genes, the only essential parts of the T-DNA are its two small (25 base pair) border repeats, which is needed for plant transformation. Agrobacterium tumefaciens STB1073 attached to the plant Crown gall disease STB1073  STB1073 Golden rice to combat vitamin A deficiency STB1073 STB1073