Lec 7 Biotechnology 2026 (PDF)

Lec 7 Biotechnology 4th Year Abdallah Makram 2026 0  Genetic engineering ‫موضوع جديد ودا نقدر نعتبره الجزء الفهم الممتع معانا هنتكلم عنه فى تلت محاضرات‬...

Lec 7 Biotechnology 4th Year Abdallah Makram 2026 0  Genetic engineering ‫موضوع جديد ودا نقدر نعتبره الجزء الفهم الممتع معانا هنتكلم عنه فى تلت محاضرات‬ Strain Gp of species with one or more characteristics that distinguish it from other members of same species Each strain is identified by name, number or letter. Example: E. coli Strain K12 ‫ساللة معينة من بكتيريا‬ Strain Improvement Targets of strain improvement Science & Technology of manipulating 1) Increase productivities ‫زودت انتاج اللى بحطه جواها‬ and improving microbial strains to 2) Regulating activity of enzymes ‫اتحكم فى االنزيمات كويس‬ enhance their metabolic capacities is 3) Introducing new genetic properties into organism by known as “Strain Improvement” Recombinant DNA technology / Genetic engineering Approaches in strain improvement 1) Mutant Selection 2) Recombination 3) Recombinant DNA Technology I. Mutation Def: It’s sudden & heritable change in traits of organism. ‫الطفرات هى تغييرات بتحصل فجاة وهى‬ Spontaneous Mutation Induced Mutation ‫نوعين نوع بيحصل فجاة لوحده من غير‬ Without any specific Ttt due to Ttt with certain agents ‫التعرض الى حاجة ونوع نتيجة التعرض‬ ‫ل ُمسبب فممكن اعمل طفرة عشان استفيد من‬ Ex: Lipase from Aspergillus japonicus MTCC 1975- mutation using ‫حاجة مثال عاوز ازود انزيم معين فى فطر‬ Makram ‫فحطوا مواد كيميائية وشافوا كل واحد هيزود‬ UV HNO2 NTG (nitrosoguanidine) NTG ‫االنزيم بنسبة قد ايه اعالهم كان‬ 127% 177% 276% higher lipase yield than parent strain. II. Recombination 1) Transformation 2) Transduction 3) Conjugation 4) Protoplast fusion ‫هنبدا اننا نتكلم عن كل نوع منهم بقى بالتفصيل فى الجزء الجاى فخلى بالك انك الزم الزم الزم تفرق بينهم كويس جدا‬ 1  1) Transformation (Liable for DNA degradation)  Genetic alteration of cell resulting from direct uptake of exogenous DNA from surroundings & taken up through CM ‫ مع الخلية‬DNA  Introduction of foreign DNA into eukaryotic cells is often called “Transfection” EXAM*********** ‫ونعمل تعديل‬ ‫فالخلية فيدخل‬  Transfection: involves opening transient pores or holes in cell plasma membrane, to allow uptake of material. ‫جوا‬  Competence: state of being able to receive foreign DNA (EXAM**********) ‫ مهم ها‬DNA from environment ‫تاخد‬ Natural transformation Artificial transformation  Efficient for linear molecules as chromosomal DNA Suitable for circular plasmid DNA but not linear DNA but not for circular plasmid molecules Chemically Competent Cells  Fragments of DNA are taken up by living recipient  CaCl2 (cation) Ttt: attachment of plasmid DNA Makram cell & recombined with recipient cell genome. ( repulsion bet DNA & membrane negative charges)  Heat-shocked in water bath opens pores of CM entry of plasmid DNA.  Examples of naturally competent bacteria: Bacillus subtilis, Streptococcus pneumonia, Electrocompetent Cells Neisseria gonorrhoeae & Haemophilus influenza. Using electroporation: create pores, genetic material enters the cells. ‫ على عكس الصناعى فى البالزميد ها مهم جدا جدا جدا دا الفكرة انى بخلى المادة الوراثية تدخل جوا الخلية طبيعى او بمادة‬DNA ‫اول نوع الطبيعى ودا بيحصل فى ال‬ 2  2) Transduction Transduction is transfer of bacterium DNA from donor to recipient Principle of transduction bacterium via virus particle ‫ركزززززززز‬ Bacterial donor DNA is incorporated into bacteriophage either through lytic or lysogenic cycle. ‫الصفحة اللى فاتت‬ Virus particle that infects bacteria is called bacteriophage or phage & phages used for transfer of DNA are called transducing phages After bacterial DNA is incorporated into phage release (Not all bacteriophages are transducing phages) EXAM**** of new phages from bact. cell infect host bacterial cell Due to high specificity of phages for cell surface receptors Phages attach to specific bacterial cell surface receptor transduction has narrowest host range of DNA transfer. & inject their DNA containing donor DNA into cytoplasm of host bacterial cell Makram Transduction doesn’t require physical contact bet. donner & receptor Transduction Transformation Depending on phage, DNA integrates into bacterial DNase resistant DNase susceptible genome, replicates in cytoplasm as a plasmid, or replicates immediately producing phage progeny Temperate phage can go both pathways (lytic cycle & lysogenic cycle) ‫ بردو جوا الخلية بس المرة دى من خالل وسيط اللى هو الفيروس فالميزة انه مش هيتكسر والفيروس اللى بيعمل كدة بسميه ترانسديوسينج‬DNA ‫الفكرة كلها انى هدخل ال‬ 3  A) Generalized transduction B) Specialized transduction Phage mistakenly packages bacterial DNA instead of own  Phage undergoes lysogeny at specific locations in bac. genome phage DNA during phage assembly infectious virus called attachment sites (temperate phage) particle containing bact. DNA  Phage genome integrates into bacterial chromosome as virus  Phage particle then attaches to bacterial cell surface receptor replication is repressed during lysogeny & injects packaged DNA into cytoplasm of bacterium.  Phage genome excises from bact. genome due to imprecise  If bacterial DNA in phage is from bacterial chromosome excision & recombination, adjacent bacterial genes are excised. DNA recombines with homologous DNA of bacterial recipient to generate stable transductions process  Unlike generalized specialized transducing phage contains both requires host recombinase, recA. phage & bacterial genes. ‫فيه االتنين مع بعض ملحومين عكس الجينيرال‬ Makram  During subsequent infection newly acquired gene is inserted into  As any of host markers can be transduced, this type of bac. genome along with phage DNA to form new round of lysogeny. transduction is termed generalized transduction  It’s independent of host homologous recombination & recA but requires phage integrase.  As this transduction mechanism is limited to genes very near original integrated prophage, it’s called specialized transduction. ‫ اى حتة منه‬DNA ‫مش جزء معين من ال‬ ‫جزء معين من البكتيريا اتنقل للفيروس‬ ‫الفيروس بيمسك فالبكتيريا ويدخل المادة الوراثية وبيبدا ياخد جزء من المادة‬ ‫الفيروس بيمسك فالبكتيريا ويدخل المادة الوراثية بردو بس هنا بيتلحم بقى فى المادة‬ ‫الوراثية بتاعته وجزء من بتاعه البكتيريا بعد كدة يعدى بكتيريا تانية يحط فيها‬ ‫ لما بيجى يتقطع بقى بيكون ميكس‬Lysogenic cycle ‫الوراثية بتاعه البكتيريا يعنى‬ Rec.A ‫المادة الوراثية دى وتندمج بال‬ ‫ بتاع البكتيريا والفيروس بعد كدة بيبدا يعدى بكتيريا تانية يصب جواها‬DNA ‫من ال‬ ‫خش الصفحة الجاية علطول نكمل الخطوات‬ ‫المادة الوراثية دى وهكذا‬ Has pure viral DNA & Bacterial DNA No pure viral DNA & Bacterial DNA 4  Steps of generalized transduction Steps of specialized transduction 1) Bac. host cell (donor cell) is first infected with phage DNA enters cytoplasm 1) After infection of donor bacterium with bacteriophage phage DNA is integrated into 2) During lytic cycle of viral replication phage DNA, along with bacterial bacterial chromosome during lysogenic cycle. chromosome is broken down into smaller pieces. 2) Due to imprecise cutting of phage DNA some 3) Some part of bact. chromosome is then packaged into one of viral capsids part of bacterial chromosome is also excised. when that is released by lysis of bact. 3) Phage containing some part of bac. chromosome 4) Transducing phage with bac. chromosome now infects second bacterium then infects new host donor DNA is incorporated (recipient bacterium) donor DNA enters cytoplasm of second bacterium. into recipient bac. during lysogenic cycle of replication. 5) In presence of host recombinase recA donor DNA recombines with Makram 4) Recipient then expresses newly acquired genetic trait. homologous DNA of bacterial recipient to generate stable transductants. Examples Generalized transducing phage Specialized transducing phage P1 phage, which can transduce E. coli DNA E. coli lambda phage transduce E. coli & integrates its DNA precisely bet. operons to numerous Gram-negative bacteria. encoding enz. respon. for galactose (gal) & biotin (bio) utilization in E. coli chromosome Note Transduction occurs when newly forming phages acquire host genes & transfer them to other bacterial cells. Generalized transduction transfer any host gene phage packaging accidentally incorporates bac. DNA instead of phage DNA Specialized transduction is due to faulty separation of prophage from bacterial chromosome new phage includes both phage and bacterial genes. 5  3) Conjugation Transfer of plasmid or other self-transmissible DNA element & Broad-host-range conjugative plasmids have been sometimes chromosomal DNA from donor cell to recipient cell via used in molecular biology to introduce recombinant direct contact usually mediated by conjugation pilus or sex pilus genes into bacterial species that are refractory to routine transformation or transduction methods Recipients of DNA transferred by conjugation are called transconjugants Process of conjugation can transfer DNA regions of hundreds to Although numerous ex. of conjugative plasmids exist, thousands of kilobases & has the broadest host range for DNA transfer conjugation involving F plasmid is the most common among methods for bacterial exchange. ‫ خلى بالك‬Transduction ‫على عكس ال‬ Occurs in & bet many species of bacteria Gram Negative as well as Gram-Positive bacteria, and even occurs between bacteria and plants Principle of conjugation Steps of conjugation Makram  Plasmid or any other genetic material is transferred from donor cell to 1) F plasmid contains tra locus results in formation of recipient cell through close physical contact pilus (pleural is pilli) on F+ cell surface.  F (fertility) plasmid of E. coli was first discovered & is one of best-studied. 2) Proteins present in pilli attach themselves on F– cell  F plasmid is present in 1 – 2 copies per cell & is very large (100 kilobases) surface pilli are resp. for making contact bet cells  E. coli harboring F plasmid are referred to as donor (F+ or male) cells & 3) Once conjugation is initiated, enz. Relaxase creates E. coli lacking F plasmid are referred to as recipient (F– or female) cells. a nick in conjugative plasmid at oriT (origin of transfer)  For transfer of F plasmid from donor to recipient, intimate contact 4) Nicked strand (called T strand) then unwinds & is between cells, resulting in mating-pair formation, is required. transferred to recipient cell in 5’-3’ direction.  Transfer of genetic material is then brought by membrane fusion of 5) Complementary strand is synthesized in both cells; two cells by action of different enzymes thus, both donor & recipient are F+. 6  4) Protoplast fusion Protoplast fusion first involves isolation of protoplasts from cells After adhesion, membranes of protoplasts fuse in some by digestion of cell wall in presence of osmotic stabilizers (EXAM) localized areas and, eventually, cytoplasm of the two protoplasts intermingle Isolation of protoplasts from gram negative organisms is generally more difficult than gram positive due to their complex cell wall. ‫ بس‬Cell membrane ‫ وخلى عندها‬Cell wall ‫خاليا وشيل منها ال‬ ‫ عشان متفرقعش خالص‬Osmotic stabilizer ‫وبحط‬ Protoplast fusion is physical phenomenon. During fusion, two or ‫ممكن الدمج دا يكون لوحده كدة او انى احط حاجة تخليه يحصل زى حاجة‬ ‫ميكانيكال تدخلهم جوا بعض او مادة كيميائية زى الكالسيوم ونترات الصوديوم‬ more protoplasts come in contact & adhere with one another ‫والبولى ايثيلين او انى استعمل شوية كهربا كمان بسيطة‬ either spontaneously or in presence of fusion inducing chemicals. Spontaneous fusion Induced fusion  Protoplasts, during isolation, often fuse  Fusion of freely isolated protoplasts from different sources with help of Makram spontaneously called spontaneous fusion fusion inducing chemicals agents is known as induced fusion.  Normally isolated protoplast don’t fuse with each other as surface of  Simple physical contact is sufficient to bring about isolated protoplast carries negative charges (-10mV to -30mV ) around spontaneous fusion among similar parental protoplasts outside of plasma membrane strong tendency in protoplast to repel each other due to their same charges. ‫نفس شحنات السطح يبعدوا بقى‬  During enzyme Ttt for isolation of protoplasts protoplasts from adjoining cells fuse through their  It needs fusion inducing chemical which reduces electronegativity of plasmodesmata to form multinucleate protoplast. isolated protoplast & allows them to fuse with each others. Methods of induced fusion Mechanical fusion Chemofusion Electrofusion isolated protoplast are brought into intimate Several chemicals has been used to induce Recently, mild electric stimulation physical contact mechanically under microscope protoplast fusion as sodium nitrate is being used to fuse protoplast using micromanipulator or perfusion micropipette ,polyethylene glycol, Calcium ions(Ca++ ). Application of fusion Fusion between non-producing strains of two species ( Streptomyces griseus & Streptomyces tenjimariensis) has yielded strain that produces Indolizomycin, a new Indolizine antibiotic. ‫فسدة‬ ‫غ جم ج‬ َّ ، ‫غل جَمهد ًة‬ ‫فإن الفرا ج‬ ُّ ‫إذا كان‬ ُ ‫الش‬ 7