Microbial Biotechnology Lecture Notes PDF

Microbial Biotechnology Dr. Eman Owis Lecturer Of Microbial Biotechnology – Mansoura Uni P h. D. G ö t t i n g e n U n i - G e r m a n y [email protected]  Microbial...

Microbial Biotechnology Dr. Eman Owis Lecturer Of Microbial Biotechnology – Mansoura Uni P h. D. G ö t t i n g e n U n i - G e r m a n y [email protected]  Microbial biotechnology Nanotechnology in Microorganisms Fermentation Genetically Modified Enzymes in different industry and Introduction associated with Biotechnology Products industry pharmatheutical industry industry & Bioethics Applications of Definition Bacteria, yeast and molds Introduction Genetic Engineering Introduction nanotechnology Production of medical Ethical aspects of Branches of Factors influencing and industrial medical and Fermenter DNA, RNA, Protein biotechnology microbial activity enzymes from pharmatheutical microorganisms biotechnology Importance of bacteria in Molecular Biology Different enzymes in History Types of fermentation the industry techniques medicine and industry Microbial Importance of Molds in biotechnology safety industry & Medicine and regulations Different techniques associated with Microbial biotechnology Genetic recombination is the exchange of information between two DNA segments. This is a common occurrence within the same species. But by artificial means, when a gene of one species is transferred to another living organism, it is called Recombinant DNA Technology. In common, this is known as Genetic Engineering.  Definition of recombinant DNA Production of a unique DNA molecule by joining together two or more DNA fragments not normally associated with each other DNA fragments are usually derived from different biological sources  Definition of recombinant DNA technology A series of procedures is used to recombine DNA segments. These new combinations of genetic material or Recombinant DNA (rDNA) molecules are introduced into the host cells, where they propagate and multiply. The technique or methodology is called Recombinant DNA technology. Several key players of recombinant DNA technique: 1. Restriction Enzymes. Cut DNA at specific sequences. e.g. EcoR1 cuts at GAATTC and BamH1 cuts at GGATCC. 2. Plasmids: independently replicating DNA circles (only circles replicate in bacteria). Foreign DNA can be inserted into a plasmid and replicated. – Plasmids for cloning carry drug resistance genes that are used for selection. 3. DNA ligase. Attaches 2 pieces of DNA together. 4. Transformation: DNA manipulated in vitro can be put back into living cells by a simple process. The transformed DNA replicates and expresses its genes. Principle of PCR The PCR technique is based on the enzymatic replication of DNA. In PCR, a short segment of DNA is amplified using primer mediated enzymes. DNA Polymerase synthesises new strands of DNA complementary to the template DNA. The DNA polymerase can add a nucleotide to the pre- existing 3’-OH group only. Therefore, a primer is required. Thus, more nucleotides are added to the 3’ prime end of the DNA polymerase. Components Of PCR Components Of PCR constitutes the following: 1.DNA Template– The DNA of interest from the sample. 2.DNA Polymerase– Taq Polymerase is used. It is thermostable and does not denature at very high temperatures. 3.Oligonucleotide Primers- These are the short stretches of single-stranded DNA complementary to the 3’ ends of sense and anti-sense strands. 4.Deoxyribonucleotide triphosphate– These provide energy for polymerization and are the building blocks for the synthesis of DNA. These are single units of bases. 5.Buffer System– Magnesium and Potassium provide optimum conditions for DNA denaturation and renaturation. It is also important for fidelity, polymerase activity, and stability. PCR Steps The PCR involves three major cyclic reactions: Denaturation Denaturation occurs when the reaction mixture is heated to 94℃ for about 0.5 to 2 minutes. This breaks the hydrogen bonds between the two strands of DNA and converts it into a single-stranded DNA. The single strands now act as a template for the production of new strands of DNA. The temperature should be provided for a longer time to ensure the separation of the two strands. Annealing The reaction temperature is lowered to 54-60℃ for around 20-40 seconds. Here, the primers bind to their complementary sequences on the template DNA. Primers are single-strand sequences of DNA or RNA around 20 to 30 bases in length. They serve as the starting point for the synthesis of DNA. The two separated strands run in the opposite direction and consequently there are two primers- a forward primer and a reverse primer. Elongation At this step, the temperature is raised to 72-80℃. The bases are added to the 3’ end of the primer by the Taq polymerase enzyme. This elongates the DNA in the 5’ to 3’ direction. The DNA polymerase adds about 1000bp/minute under optimum conditions. Taq Polymerase can tolerate very high temperatures. It attaches to the primer and adds DNA bases to the single strand. As a result, a double-stranded DNA molecule is obtained. These three steps are repeated 20-40 times in order to obtain a number of sequences of DNA of interest in a very short time period. Production of Insulin