Biotechnology Practical Course 2025 - MUST University PDF

Biotechnology Practical course Faculty of Pharmacy MUST University 2025 Dr/ Mohamed Hatem 0122 910 99 83 1  Introduction of biotechnology  biotechnology : application that uses biological systems , living organisms to make or modify pr...

Biotechnology Practical course Faculty of Pharmacy MUST University 2025 Dr/ Mohamed Hatem 0122 910 99 83 1  Introduction of biotechnology  biotechnology : application that uses biological systems , living organisms to make or modify products for specific use 1.Medical Biotechnology :  Medic use of living cells to better the health of humans  The technique is used to produce pharmaceutical drugs or to combat diseases.  The study of DNA (Deoxyribonucleic acid) is used to manipulate genetic makeup of cells to increase production of beneficial human cell products ( ex: insulin )  Examples of Medical Biotechnology : ( Vaccines, Antibiotics ) 2. Agricultural biotechnology :  Agricultural biotechnology focuses on developing genetically modified plants to ( increase crop yields or introduce characteristics to those plants ) that enable them growing in regions of stress factor, weather, and pests  Examples : Pest Resistant Crops 3. Industrial Biotechnology :  refers to the use of microorganisms or biological substances such as ( enzymes ) to perform industrial or manufacturing processes.  Applying the techniques to processes including ( paper, chemical manufacturing, and textile )  Examples :  Biocatalysts : living systems to speed up chemical reactions ( such as enzymes )  Fermentation : is a metabolic process that produces chemical changes in organic substrates by the action of enzymes. 4. Environmental biotechnology :  related to the use of microorganisms and their products in ( prevention, treatment, and monitoring of environmental pollution , wastes biotreatment ).  Environmental engineering : introduce nutrients to stimulate the activity of bacteria that already exists in the soil at a waste site or add new bacteria to the soil.  The bacteria help ( digesting the waste at the site and turning it into harmless byproducts ) Examples : Bioremediation : is the use of microbial species to clean up soil and groundwater 2  Growth Curve : 1- Lag Phase ----- Prepares Bacteria for Exponential Growth bacteria , adapt themselves to growth conditions 2- Log phase ( = exponential phase) ------ characterized by cell doubling 3- Stationary phase ----- growth-limiting factor such as the depletion of an essential nutrient, and formation of an inhibitory product. Stationary phase results from a situation in which growth rate and death rate are equal. 4- Death phase ( =decline phase): bacteria die.  colony forming units: - Enumeration by colony forming units (CFU) provides a direct measurement of bacterial cell counts.  Optical density : - It gives approximation of bacterial cell count measured using a spectrophotometer. - The optical density is a measure of absorbance of light particles that pass through 1cm of culture and detected by a photodiode sensor. 3  Maintenance and preservation of pure cultures : - pure cultures are transferred periodically onto ( fresh medium ) , a procedure known as Subculturing ----- allows continuous growth and viability of microorganisms - Refrigeration : stored at 0- 4°C either in refrigerators or in cold- rooms. - This method is applied for short duration (2- 3 weeks for bacteria) and ( 3- 4 months for fungi ) - Paraffin Method/ Mineral Oil Storage : simple and the most economical method of maintaining pure cultures of bacteria and fungi. - Cryopreservation : (i.e., freezing in liquid nitrogen at - 196°C) helps survival of pure cultures for long storage times. The process invariably relies on the use of - Cryoprotectant ---- which protect Microorganisms from damage due to formation of ice crystals during freezing ( such as : glycerol , dimethyl sulfoxide (DMSO), polyethylene glycol ) - Lyophilization ( = freeze drying ) : culture is rapidly frozen at a very low temperature (- 30 to - 70°C) and then dehydrated by vacuum. microbes go into dormant state for years. - Preservation on Paper : used for Spore forming fungi , actinomycetes can be preserved on pieces of sterile filter paper and stored at room temperature - Preservation on Beads : Porcelain beads are autoclaved in screw cap glass vials. The sterile beads are transferred to sterile Petri dish and inoculated with the cell suspension. - Silica gel : used to preserve conidia and Mycellium ( of fungi ) 4  Principal Steps Of Bioengineered Biotechnology Process : 1- Upstream processing: preparation of microorganism and raw materials required for the microorganism to grow and produce the desired product 2- Fermentation and transformation: growth of microorganism in a large bioreactor ( >100 liters) with (biotransformation) of a desired compound ( example, an antibiotic , amino acid , protein ) 3- Downstream processing: recovery and purification of the desired compound from either medium or cell mass  Selection Of Industrially Important Microorganisms : - increase the growth rate of desired species of microorganism , either by : 1- discouraging growth of other unwanted microorganisms ---- ( Enrichment medium ) 2- killing unwanted microorganism ----- ( Selective medium )  Criteria considered while selecting desired organism : 1- Nutritional characteristics : culture medium must be cheap and readily available 2- Optimum temperature : If the microorganism is capable of growing at a higher temperature above 40 ° C, it reduce the costs involved in the cooling 3- Suitability: The microorganism should be suitable for the industrial process and not react with fermentation vessel or equipment. 4- Stability: The microorganism should be genetically stable 5- Yield: should provide high yield of product per unit time 6- Downstream Processing: Easy product recovery and purification of the product from the medium 7- Toxicity: The organism or its products should not be toxic. 5  Methods used for selection and isolation of microorganisms : 1- Chemical Methods : A- Enrichment Culture : - It is used to isolate those microorganisms, which are present in small numbers or in mixed culture with other organisms - enrichment culture incorporating a specific nutrient ( ex: carbon or nitrogen source ) B- Use of minimal nutrient : - Many microorganisms require dilute medium components C- Use of Inhibitors : - ( ex: dyes , bile salts , heavy metals and antibiotics ) used for selecting groups of bacteria - Crystal violet ----- inhibits all Gram-positive bacteria, except enteric bacteria (= which survive in bile salts ). - MacConkey agar ----- permit only ( Enteric bacteria ) to grow 2- Physical Methods : A- Heat : used to isolate the endospore forming bacteria , at a high temperature (80 ° C) - Heating to this temperature kills the vegetative cells of other microorganisms B- Incubation Temperature : Microorganisms can be differentiated three categories - Thermophiles --- optimum 50 c - Mesophiles --- optimum 37 c - Psychrophiles ---- optimum 15 c C- PH : microorganisms prefer to live in a particular pH range 3- Biological Methods : - An excellent example is using ( chick embryo ) for growth and purification of selective groups of viruses - N.B : ( The aim of all these methods is to isolate pure cultures of microorganisms ) - Pure culture : is a culture containing single kind of organism free from other organisms. 6  Screening od micro-organisms :  Primary screening : - isolation and identification of industrial organism from mixed population ( using simple techniques natural source , e.g soil ) - it is based on certain biochemical characters of microorganism Example : A- Primary Screening for Organic Acid/ Amine Producers : 1. Source --- ( soil, milk, milk product ) 2. serially diluted soil sample is plated and Spread onto agar medium 3. Incubate at optimum temp 4. isolated colony are selected 5. Isolated in ( pH indicating dye , such as Neutral red (Pink to yellow) or Bromothymol blue (Yellow to blue)). 6. Change in color of media (acid or amine) representing an acidic or alkaline reaction 7.calcium carbonate is added B- Primary Screening of Antibiotic Producers : 1. The crowded plate technique ---- is the simplest screening technique employed in isolation of antibiotic - It consists of series of dilution of the raw material for the antibiotic , followed by spreading on agar plates 7  Secondary screening : - It is important for sorting out commercially important microorganisms from a variety of microorganisms obtained during primary screening.  biotechnological process is three stages: - Stage I : Upstream processing : which involves ( preparation of liquid medium, separation of inhibitory chemicals , sterilization, air purification ) - Stage II: Fermentation : which involves ( conversion of substrates to desired product ) - Stage III: Downstream processing : which involves ( separation of cells from fermentation broth, purification and concentration of desired product ).  Fermenter : is a vessel for growth of microorganisms which not permitting contamination, enables conditions necessary for maximal production of desired products ( optimal pH, temperature, oxygen ) and other environmental conditions. - Fermenters are also known as ----- ( bioreactors ). - Fermenters may be ----- ( liquid = submerged or solid = surface ) culture medium - Most fermenters used in industry are submerged type ---- because saves space and is more amenable to engineering control and design.  Fermenter/ Bioreactor components :  Typical fermenter is upright closed cylindrical tank fitted with : - four or more baffles attached to side wall - water jacket or coil for heating and/or cooling - device for forcible aeration ( known as ---- Sparger ) - mechanical agitator ----- pair or more impellers 8   Modern fermenter : usually integrated with computers for efficient continuously monitoring, controlling or recording of : ( pH , oxidation-reduction potential , dissolved oxygenand CO2, chemical components of broth )  Fermenter Body Construction :  Material used for the construction of fermenter body differs according to scale : (small , pilot and industrial scale)  essential features of construction material is ---- ( non-toxic and corrosion proof )  fermenter can be as small as 1 - 20 liters -- --- in laboratory-scale  fermenters and range from 100,000 - 500,000 liters ---- for production industrial scale 1- Small scale fermenter : - Glass vessel : is generally made up of borosilicate glass. - Glass vessels are of two types: * Type I –glass ---- round or flat glass bottom with top plate , can be sterilized by autoclaving. - The largest type I glass vessels have a diameter 60cm. *Type II –glass : flat bottom and with top and bottom stainless-steel plate. - This type is used in ( in situ sterilization ) , largest vessel has diameter 30cm. 2- Pilot and large-scale fermenter : - Stainless steel or have stainless steel cladding ------- to limit corrosion. - steels containing less than 4% chromium ----- as alloys containing more than 4% chromium are classified as stainless steels  Aeration in Fermenter : - The purpose of aeration ---- provide oxygen for growth and metabolites production in aerobic organisms. - air used is sterile - Example : production of citric acid ----- broth is entirely saturated with oxygen 9  Agitation : - Agitation provides ----- mixing of nutrient with micro-organisms - In large-scale operations ------ aeration is maintained by agitator - Agitators attached to ( impellers ) ---- They help to distribute air as fine bubbles, mix organisms uniformly as well as uniform temperature - The viscosity of broth may alter fermentation rate ---- ( such as : filamentous fungi and actinomycetes )  Temperature Control in Fermentor : 1- small = laboratory fermenters ----- by immersing tank in water bath 2- medium size = pilot fermenter ---- jacket of cold water circulating outside the tank 3- large = Industrial fermenters ------- circulating refrigerated water in pipes within fermenter or outside  Foam Production and Control : - Foams are dispersions of gas in liquid. - It occur as a ------ result of agitation and aeration. *Why foam has undesirable : 1- substantial headspace is left in industrial fermentations. 2- oxygen transfer rate is reduced. 3- foam escapes contamination and may cause wetting filters and other portions of fermenter. 4- Organic nutrients or inorganic ions give complex organic compounds 5- fermentation product may be removed 6- Loss of microorganisms ---- leading to reduced yields.  Foam control : - Materials that yield foam , ( such as : proteins, peptides, synthetic detergents, soaps, and saponin) ----- They lower surface tension  Foams are controlled either by chemical or mechanical methods : 1- Chemicals controlling methods : classified into : * antifoams ---- which are added in medium to prevent foam formation * defoamers ---- which are added to knock down foams once these are formed. 10   Antifoams should ideally have following properties: 1- non-toxic to microorganisms and higher animals 2- no effect on taste and odor 3- be autoclavable. 4- not be metabolized by microorganisms 5- not impair oxygen transfer. 6- be active in small concentrations, cheap  chemical antifoams Examples : Category Example Remarks 1- Natural oils Peanut oil, Not very efficient Used as ; and fats soybean oil may be metabolized 2- Alcohols Sorbitan Not very efficient may be toxic or metabolized 3- Sorbitan Sorbitan Span 20 is active in extremely monolaureate small amounts. derivatives 4- Polyethers P400, P1200, Active, but varies with P2000 fermentation 5- Silicones Silicones Very active; inert, low toxity; expensive. 2- Mechanical defoamers : - Mechanical defoamers have various designs - They act by physically dispersing the foams and breaking them up.  Process Control in a Fermenter : 1- pH measurement and control : - pH is important factor that control microbial growth - control of pH is achieved by ---- ( natural buffers in the medium OR phosphates and calcium carbonate ) - Sterilizable pH probes have become available, to measure PH in the fermenter 11  2- Carbon dioxide measurement : - A sensitive sensor translates CO2 levels to a gauge or record 3- Oxygen determination and control : 4- Pressure : - A positive pressure helps eliminate contamination and maintenance of aeration. - Pressure may be determined with aid of ----- manometer. 5- Computer control : # ANAEROBIC BATCH FERMENTORS : they have the following characters : 1 Air sparging is absent --- ( as oxygen is not required ). 2 Agitation is aimed only for ---- (distribution of organisms, nutrients and temperature ) 3 The medium is introduced into fermenter while hot ---- to prevent absorption of O2 Gas 4 The fermenter is filled as much as possible ---- in order to avoid airspace 5 Some low redox compounds ( ex: cysteine ) - Two strictly anaerobic fermentation processes include : 1- acetone-butanol fermentation --- by (Clostridium acetobutylicum) 2- anti-tetanus toxoid production ---- by (Clostridium tetani). - Example of a micro-aerophilic fermentation which requires only a small amount of oxygen: - ------ lactic acid production  FERMENTOR OPERATIONS : 1- Batch fermenter : 12 2- Continuous fermenters : - It differs only in there is provision for : a- inlet of medium and b- outlet of broth 3- Tubular fermenters : - The tubular fermenter was so named because ---- it resembled a tube. - tubular fermenters are continuous unstirred fermenters - Reactants enter at one end and leave from the other - Due to the absence of mixing ----- there is a gradual fall in the substrate concentration between the entry point and the outlet while there is increase in product concentration 4- Fluidized bed fermenter : - The microorganisms are kept in suspension by the medium flow rate whose force t balances gravitational force. - If flow were lower ---- the bed would remain ‘fixed’ - If flow rate was higher ----- ‘elutriation’ would occur (= particles will be washed away from the tube ) 13  Extraction of fermentation products : The following factors considered during extraction : 1- value of final product 2- degree of purity required in final product 3- chemical and physical properties of the product 4- location of the product in mixture ---- ( whether free within medium or cell- bound ) 5- properties of impurities 6- cost-effectiveness of isolation procedures.  Downstream processing divided into four stages : 1- Removal of insoluble (solids) : - Example: ( separation of cells , cell debris from fermentation broth ) - operations to achieve this include : - Filtration - Centrifugation --- but it is not widely used - Coagulation ( = cohesion of colloids into small flocs), - Sedimentation and flocculation 2- product isolation : - separation of desired product from other components by : ( extraction, evaporation, adsorption, ultrafiltration and precipitation ) - selective isolation of some industrial Products by : ( adding inorganic salts and membrane filtration ) 3- product purification : - separation of products from contaminants - operation include --- ( chromatography, crystallization , membrane filtration , decolorization and fractional precipitation ) 4- product polishing : - Is final processing step which ends with packaging of product - Include ---- ( lyophilization, Drying , Crystallization and sterilization ) 14  Molecular biotechnology : It is scientific discipline that is based on ability to transfer specific units of genetic information from one organism to another. This relies on techniques of genetic engineering (recombinant DNA technology). The objective of recombinant DNA technology is ------ create a useful product or commercial process Molecular biotechnology can contribute benefits to humanity. It can: Molecular biotechnology Provide opportunities to : 1- accurately diagnosis , prevent, or cure of infectious and genetic diseases ) 2- increase crop yields by creating plants resistant to insect predation, fungal and viral diseases 3- Develop microorganisms that produce chemicals, antibiotics, polymers, amino acids, enzymes, and various food additives 4- Develop livestock and animals that have genetically enhanced characters 15  Recombinant DNA technology : It is also called ( gene cloning or molecular cloning ) is a general term that encompasses number of experimental protocols leading to ( transfer of genetic information (DNA) from one organism to another ) (cloned DNA = insert DNA = target DNA ) from donor organism is extracted, enzymatically cleaved and joined (ligated) to another DNA entity ( cloning vector ) to form recombined DNA molecule ( cloning vector = insert DNA construct ). The introduction of DNA into a bacterial host cell is called ------ ( transformation ). Those host cells that take up DNA construct is called ------ (transformed cells) DNA construct can create protein product in host cell.  GENE CLONING : - Gene : it is a segment of DNA molecule that consists of about 1000 nucleotides - Genetic engineering or recombinant DNA technology has made it possible to isolate a desired gene from large complex genome - The specific gene is cut off, ligated into a suitable vector which allows the gene to be amplified enormously in the host cell. - The process is referred to as gene cloning - Gene cloning involves taking a piece of DNA from organism where it naturally occurs and putting it into a cloning host such as the bacterium ( Escherichia coli ).  Isolation and purification of genomic : 16   PCR Steps : 1- Denaturation : - Denaturation occurs when the reaction mixture is heated to 94℃ for about 0.5 to 2 minutes. - This breaks hydrogen bonds between the two strands of DNA and converts it into single-stranded DNA. 2- Annealing : - The reaction temperature is lowered to 45-72℃ - primers are single-strand sequences of DNA or RNA , bind to their complementary sequences o n the template DNA. 3- Elongation : - At this step, the temperature is raised to 72℃. - The bases are added to the 3’ end of the primer by the Taq polymerase enzyme. - Taq Polymerase can tolerate very high temperatures. - It attaches to the primer and adds DNA bases to the single strand. As a result, double-stranded DNA molecule is obtained.  Digestion of source DNA with restriction nuclease : - For cloning, desired gene must be inserted into ----- plasmid or other vectors To insert the gene in suitable vector, two points needs : 1- The size of DNA fragments to be inserted into vector N.B : gigantic genomic DNA is divided into short fragments so easily be inserted - Fragmentation of DNA is accomplished by restriction nuclease enzymes 2- The type of restriction nuclease used to cut DNA molecule into fragments - There are many restriction enzymes isolated from different microorganisms. - Each restriction enzymes recognizes and cleaves double stranded DNA molecule into smaller fragments at specific recognition or cutting site ---- determined by nucleotide sequence 17   INSERTION OF DIGESTED DNA INTO SUITABLE VECTOR :  DNA source ---- is digested with a particular restriction nuclease, Then , inserted into suitable vector. Then , enable it to be replicated under suitable conditions  Vector DNA ---- is also digested with the same restriction enzyme  foreign DNA is inserted and ligated in the vector DNA by DNA ligase Then , recombinant vector insert into host suitable for multiplication Then , isolation of the right clone. ----- by transformation methods. including ( head shock and electroporation ) DETECTION OF CLONE CONTAINING THE DESIRED GENE ( Confirmation by sequencing and PCR )  Vaccine : - vaccination protects a recipient from pathogens by establishing an immunological resistance - injected or oral vaccine induces the host to generate antibodies against disease-causing organism - Therefore, infectious agent is inactivated (neutralized, or killed) - First vaccination was documented by Edward Jenner in 1798 - The success of vaccination relies on ( immune response and long-lasting memory ). - Vaccination is also called ( active immunization ) , because the host’s immune system is activated to respond to “infection” by itself 18  Characters of Ideal vaccine : - 100 % efficient in all individuals of any age - lifelong protection after single administration - Does not evoke an adverse reaction - stable under various conditions (temperature, light, transportation) - easy to administer, preferably orally - available in unlimited quantities - cheap  Type of vaccine : 19 1- Live Attenuated Vaccine : - It uses non-pathogenic form of infectious organism - limited ability to replicate in host ----- therefore , unable to cause disease. - attenuated microbe ---- simulates an infection without causing disease - Attenuation done by growing microbe in abnormal conditions ( ex: Low temperature ) or modifying genes needed for replication - Attenuated virus vaccines are obtained by ---- ( growing virus in embryonated eggs or tissue culture cells at low temperatures (25°C - 34°C) - These viruses called ( low-temperature mutants ) ----- grow slowly and less virulent - The earliest vaccines were live attenuated organisms : e.g. 1- smallpox vaccine by Jenner 2- rabies vaccine by Pasteur. - Live vaccines have complex upstream cultivation but , minimal downstream processing * Advantage : - induces memory. - Most vaccines require one exposure - delivered by alternative routes. For example, polio vaccine orally or parentral *Disadvantage : - NOT safely given to people who are immunocompromised - NOT given to women during pregnancies. live attenuated examples polio vaccine developed by Albert Sabin and measles, mumps, and rubella (MMR) 2- Inactivated Vaccine ( = Killed ) : - are made by killing or inactivating pathogen by ( heat, or chemical means ). - pathogen can no longer replicate within the host but can generate immune responses. - example of inactivated vaccine is polio vaccine developed by Jonas Salk ---- by formaldehyde treatment of poliovirus *Advantage : - pathogen is no longer capable of replicating within host. - It is easily phagocytosed and presented to Th cells. - broad immune response. - no disease in host. - relatively heat stable. 20 *Disadvantage : short-lived – requires booster vaccinations. Many pathogens have endotoxins that are not removed , so may cause serious side effects. 3- Toxoid vaccine (inactive toxin) : - Toxoid vaccines (e.g., tetanus or diphtheria) - The toxin is rendered harmless to form (toxoid) and used as antigen to elicit immunity *advantage : - safe , because they cannot cause disease *Disadvantage : - required multiple boosters - Less effective immune response 4- DNA Vaccines : - DNA coding for an immunogenic protein of a pathogen can be inserted *Advantage : - Safe in case of immunosuppressed individuals. - Less side effects *Disadvantage : - Immune responses are primarily humoral only 5- Subunit vaccine : - Molecular components of bacteria or viruses can be used as vaccines. - These include capsular polysaccharides and surface proteins of bacteria and viral envelope - Example : hepatitis B virus (HBV) vaccine consists of HBsAg, - Capsular polysaccharides are used as vaccines for Streptococcus pneumonia. Neisseria meningitides and Haemophiles influenzae B 21  Molecular diagnostics : Many of these detection procedures require the growth in culture and then analysis Although tests are effective and specific, but they are slow and costly. if the pathogenic organism cannot be cultivated, the opportunity to detect organism is limited. For example, Chlamydia trachomatis, causes a sexually transmitted disease Clinical diagnosis of chlamydial infection is difficult, Frequently give false-negative results To overcome : molecular diagnostic procedures using either immunological or DNA detection methods ( ex: microarray assays, real-time PCR )  Gene therapy : Gene therapy is use of nucleic acids as a pharmaceutical agent to treat disease Gene therapy targets defected genes only, without affecting normal genes Several strategies can be used for gene transfer. Direct injection of vector/DNA complexes into the bloodstream is often characterized by low gene expression and adversely affect the function and health of normal tissues Gene transfer is achieved by direct application of the vector (virus, plasmid) for efficient gene expression Gene therapy can be classified into : 1- viral gene therapy OR 2- nonviral gene therapy  Type of gene therapy: 1- In Vivo Gene Therapy : - Therapeutic nucleic acids are directly delivered inside a patient’s body using a viral or non-viral delivery system. 2- Ex Vivo Gene Therapy : - It refers to genetic manipulation of cells outside the body  N.B : In vivo gene therapy is not as successful as ex vivo gene therapy for cancer primarily because of the increased risk of unprecedented effects 22  Non-Viral Delivery Systems : 1- Nanoparticles : - NPs are nanometer sized (