lOMoARcPSD|22115400 Genetic Engineering Chemical and Biotechnological Process Engineering (Universitat Politècnica de Catalunya) Studocu is not sponsored or endorsed by any college or university Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT genetic engineering BT/PI Lesson 8 GENETIC ENGINEERING - Introduction - General steps for the introduction of a new gene and its expression - Some examples and problems that may arise Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT genetic engineering BT/PI introduction What is genetic engineering? Genetic engineering comprises a set of techniques that allow direct manipulation of the genetic material (DNA) in order to eliminate, modify or introduce novel characteristics into an organism. Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT genetic engineering BT/PI introduction Selective breeding is actually a way to select genes… …but genetic engineering allows to obtain the desired results in a more direct and controlled way by acting on DNA AND allows to cross the limits imposed by species Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT genetic engineering BT/PI Lesson 8 GENETIC ENGINEERING - Introduction - General steps for the introduction of a new gene and its expression - Some examples and problems that may arise Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT genetic engineering BT/PI introduction and expression of a new gene Example: CHYMOSIN (RENNIN) natural source: calf (4th stomach) limiting! Could it be obtained by genetic engineering? Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT genetic engineering BT/PI introduction and expression of a new gene general steps: A) Obtaining the DNA fragments B) Obtaining recombinant DNA C) Transformation D) Cloning and expression of the gene Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT genetic engineering BT/PI obtaining the DNA fragments A) Obtaining the DNA fragments: RESTRICTION ENZYMES (1970) Restriction enzymes or restriction endonucleases are found in bacteria and archaea and provide a defense mechanism against invading viruses by cleaving foreign DNA. (host DNA is protected by a modification enzyme that modifies the prokaryotic DNA and blocks cleavage) Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT BT/PI genetic engineering obtaining of DNA fragments → restriction enzymes Recognition site or restriction site: Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA. Usually, recognition sites have between 4 and 8 base pairs and many of them are palindromic. HaeIII - Haemophilus aegyptius blunt ends EcoRI - Escherichia coli sticky ends Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT genetic engineering BT/PI obtaining recombinant DNA B) Obtaining recombinant DNA: JOINING OF DNA FRAGMENTS 1) Joining of DNA fragments with sticky ends 2) Joining of DNA fragments with blunt ends Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT BT/PI genetic engineering obtaining recombinant DNA → joining of DNA fragments 1) Joining of DNA fragments with sticky ends Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT BT/PI genetic engineering obtaining recombinant DNA → joining of DNA fragments Joining of DNA fragments with sticky ends complementary base pairing is not enough! ligase Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT BT/PI genetic engineering obtaining recombinant DNA → joining of DNA fragments 2) Joining of DNA fragments with blunt ends … … ligase Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT genetic engineering BT/PI transformation C) Transformation: Genetic alteration of a cell by the direct uptake and expression of DNA from its surroundings(→ transformed cells) IT IS NECESSARY TO CONSIDER: 1) VECTORS 2) HOST SYSTEM 3) DETECTION AND SELECTION OF TRANSFORMED CELLS Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT BT/PI genetic engineering transformation → vectors 1) VECTORS DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated. TYPES: Plasmids, viral vectors, cosmids ADVANTAGES: - Easier entry of DNA - Greater stability - They facilitate that this DNA functions and replicate - Easier identification of transformed cells Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT BT/PI genetic engineering transformation → vectors Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT BT/PI genetic engineering transformation → vectors Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT genetic engineering transformation → hosts BT/PI 2) HOST SYSTEMS - Bacteria: Escherichia coli, Bacillus, Pseudomonas - Yeasts: Saccharomyces - Algae, plants, mammalian cells Requirements (e.g. E. coli) 1. Recipient bacteria must be in a state of competence to be able to allow the entry of DNA (methods: CaCl2, electroporation) 2. Not present functional restriction enzymes 3. In the case of antibiotic resistance selection, the host has to be sensitive to the antibiotic for which the vector carries a gene of resistance 4. Not suppose any danger to humans or other organisms Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT BT/PI genetic engineering transformation → detection & selection of transformed cells 3) DETECTION AND SELECTION OF TRANSFORMED CELLS Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT BT/PI genetic engineering transformation → detection & selection of transformed cells - auxotrophic markers how? - color detection - antibiotic resistance - etc. Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT BT/PI genetic engineering transformation → detection & selection of transformed cells Selection by antibiotic resistance Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT genetic engineering BT/PI cloning and expression of the gene D) Cloning and expression of the gene: Some possible problems: - Low polypeptide stability in the new environment - Product toxicity for the host cell - etc. Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT genetic engineering BT/PI Lesson 8 GENETIC ENGINEERING - Introduction - General steps for the introduction of a new gene and its expression - Some examples and problems that may arise Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT genetic engineering BT/PI examples and problems 1) CHYMOSIN - E. coli In the cells of the stomach of the calves the chymosin is synthesized as an inactive precursor. preprochymosin hydrolysis in acidic environment (stomach) chymosin The gene had to be cloned and expressed in E. coli in the form of mature chymosin. Recombinant chymosin is present in the cheese market since 1990. It is called fermentation-produced chymosin (FPC) and has exactly the same amino acid sequence as calf chymosin. Nowadays, FPC is mainly produced by fungi (Aspergillus niger or Kluyveromyces lactis). Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT genetic engineering BT/PI introduction 2) HUMAN INSULIN - E. coli Insulin is a hormone of protein nature that is used for the treatment of diabetes. Insulin from animal sources (from bovine or porcine pancreas) produces several side effects and is not as effective. Since 1982 recombinant human insulin is obtained from genetically modified microorganisms. Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT genetic engineering BT/PI examples and problems but it was not an easy task… - preproinsulin - 1 intron Downloaded by Maribel Manes (mabellaganmanes@gmail.com) lOMoARcPSD|22115400 BT genetic engineering BT/PI examples and problems 3) SUBTILISIN - Bacillus subtilis Subtilisin, a protease initially obtained from Bacillus subtilis, is commonly included in the composition of laundry detergents and other products. Site-directed mutagenesis techniques have made it possible to improve subtilisin properties (protein-engineered subtilisins). wild-type enzyme resistance to bleach resistance to temperature SITE-DIRECTED MUTAGENESIS Downloaded by Maribel Manes (mabellaganmanes@gmail.com)
