APBI305 Lecture 1.pdf

Genetic Engineering II [APBI 305] Basic Concepts of Genetic Engineering By Dr. Arwa Kohela Assistant Professor of Molecular Biology Course specification Major Status Code Title Credit hours...

Genetic Engineering II [APBI 305] Basic Concepts of Genetic Engineering By Dr. Arwa Kohela Assistant Professor of Molecular Biology Course specification Major Status Code Title Credit hours Applied Core APBI305 Genetic 2 CH lecture biotechnology Engineering II 4 CH lab (4 contact) Course prerequisites Level hrs) Semester APBI303 (Genetic Engineering I) Third level Spring Course completion requirements: Grading: Attending >25% of course classes Final theoretical exam: 40% Passing the final theoretical exam Final practical exam: 20% Oral exam (project): 10% Midterm term: 10% Coursework: 20% Total: 100% APBI 305 Course aim The aim of this course is to provide the students with the knowledge and hands- on skills on the advanced tools of genetic engineering and their applications in the different sectors of biotechnology. APBI 305 Course contents 1. Basic concepts of genetic engineering 2. Transgenic animals part I 3. Transgenic animals part II 4. Genome editing part I 5. Genome editing part II 6. Genome editing part III 7. Genome editing part IV 8. Human gene therapy 9. Transgenic plants part I 10. Transgenic plants part II APBI 305 Course competencies 1. Integrate knowledge and procedures to produce and identify genetically modified organisms (GMOs). 2. Recognize the role of recombinant DNA in manipulating organisms. 3. Apply different molecular tools to study human diseases in model organisms. 4. Develop biotechnological techniques to design novel and personalized therapies for human disorders. APBI 305 Course learning outcomes A= Cognitive LOs B= Psychomotor LOs C= Affective LOs A1. Recognize the concepts of gene editing and their applications in different industries. A A2. Explain the various molecular tools used to manipulate genomes. A3. Describe the methods used to deliver transgenes into living organisms. B1. Conduct experimental procedures to clone mammalian genes into E. coli. B B2. Use molecular tools to genotype transgenic animals and quantify gene expression. B3. Design in silico procedures to manipulate genomes using CRISPR/Cas9 gene editing tool. C C1. Describe the benefits and limitations of genome editing. APBI 305 References Textbooks Molecular biology of the cell (6th edition) by B Alberts, A Johnson, J Lewis, D Morgan, M Raff, K Roberts and P Walter. Garland Publishing, New York (2015) Genome Engineering via CRISPR-Cas9 System by Vijai Singh and Pawan K. Dhar. ISBN 978-0-12- 818140-9 (2020) Transgenic Animals by Louis-Marie Houdebine (2022). ISBN: 9781000448436, 1000448436 Articles Hyeonhui, K, Minki, K, Sun-Kyoung, I, and Sungsoon, F. Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes. Lab Anim Res. (2018) 34:147–59. doi: 10.5625/lar.2018.34.4.147 Uddin F, Rudin CM, Sen T. CRISPR gene therapy: applications, limitations, and implications for the future. Front Oncol. 2020;10:1387. doi:10.3389/fonc.2020.01387 Lino CA, Harper JC, Carney JP, Timlin JA. Delivering CRISPR: a review of the challenges and approaches. Drug Deliv. 2018;25(1):1234–57 APBI 305 Human Genome Every cell carries the same genetic information representing the DNA genome. The genome of Homo sapiens is stored in 23 chromosome pairs located in the nucleus plus the mitochondrial DNA. DNA is a double stranded molecule that carries the genetic information essential for the development and functioning of most living organisms. APBI 305 DNA Structure DNA is a double stranded molecule that carries the genetic information essential for the development and functioning of most living organisms. DNA is composed of 2 long strands running in opposite directions (antiparallel). Each strand is a polymer composed of nucleotide units linked together along the chain. APBI 305 Nucleotide Structure A nucleotide is composed of a nitrogenous base, a deoxyribose sugar and a phosphate group. Types of Nucleotides: - Purines: Adenine & Guanine - Pyrimidines: Cytosine & Thymine Where Adenine pairs with Thymine by a double H-bond and Guanine pairs with Cytosine by a triple H-bond. The sugar and phosphate groups are linked by ester bonds and constitute the molecule backbone. APBI 305 Nucleotide Structure APBI 305 DNA Replication DNA is unwound by helicase by breaking hydrogen bonds between the 2 stands. Unwinding in done at multiple locations forming replication bubbles. To prevent re-annealing of the strands single stranded binding proteins (SSBs) bind to the exposed DNA. A primer is added to complement the parental strand by the primase enzyme. DNA polymerase III catalyzes the addition of nucleotides in the 5' to 3' direction. APBI 305 DNA Replication The leading strand undergoes continuous replication since its template has a 3’ to a 5’ directionality. However, the lagging strand undergoes a discontinuous replication as its template strand has a 5’ to 3’ directionality. The primase and polymerase enzymes must therefore work in a reverse direction causing multiple breaks during synthesis of the lagging strand. The resultant small fragments of DNA are synthesized on the lagging strand are known as Okazaki fragments, which are joined together by DNA ligase. APBI 305 DNA Replication APBI 305 The DNA Code DNA contains segments of information called genes, either protein coding or non-coding. The estimated number of protein-coding genes in human is 25,000 genes. Genes are composed of 3 letter units called codons. DNA copies its information into and mRNA molecule that in a process called transcription. mRNA is composed of codons that code for amino acids that when linked by peptide bonds form a protein in a process called translation. APBI 305 The Central Dogma of Molecular Biology APBI 305 Gene Expression The biological information stored in genes is utilized by a complex system of enzymes and proteins in a process termed gene expression. Transcriptome: collection of RNA molecules in a certain cell type and a particular time. Proteome: cell's repertoire of proteins that specifies the biochemical reactions carried out by a particular cell. APBI 305 RNA Coding mRNA: Encoded by protein-coding genes and translated by the ribosomal cell machinery into proteins. Non-coding RNAs: Play mainly regulatory roles in the cells. The most prominent examples are rRNA and tRNA, which are both involved in the translation process. APBI 305 Transcription Transcription is the process by which a particular segment of DNA is copied into an RNA molecule using a complex system of proteins and transcription factors. Transcription is divided into: 1. Initiation: Binding of RNA polymerase to the promoter region in target gene. 2. Elongation: The sequential addition of nucleotides in the 5’ to 3’ position. 3. Termination: Upon recognition of termination signals, RNA polymerase dissociated from the coding strand the nascent RNA molecule is released. APBI 305 Transcription APBI 305 Post-transcriptional Modification A process by which eukaryotic cells convert the primary RNA transcript into mature RNA. e.g. pre-mRNA modification Ø splicing of introns Ø poly A tail Ø 7-methylguanosine 5' cap APBI 305 Translation The process by which ribosome create polypeptides using the information carried on an mRNA molecule. Polypeptides are later folded and post-translationally modified to carry out a specific function in the cell. APBI 305 Translation The process of translation is done as follows: Initiator tRNA binds to the start codon on the mRNA. Large and small ribosomal subunits join to form a functional ribosome and assemble around the mRNA. The tRNA adds the first amino acid to start the peptide chain. The ribosome moves to the next mRNA codon to continue the process. Amino acids are linked by peptide bonds. When a stop codon is reached the ribosome and mRNA are dissociated and the polypeptide chain is released. APBI 305 Translation APBI 305 Post-translational Modification Chemical modification of proteins during or after protein synthesis that regulates protein function, activity and location. e.g. phophorylation, glucosylation, ubiquitniation, methylation, acetylation, proteolysis. APBI 305 Gene Expression APBI 305 DNA Cloning A technique that allows selecting and amplifying a specific DNA sequence. Applications: Ø Amplification and propagation of DNA. Ø Sequencing of genomes Ø Identification of mutations Ø Engineering DNA for specific purposes. In vitro cloning: PCR In vivo cloning: Cell-based APBI 305 Polymerase Chain Reaction A technique used to amplify a specific DNA sequence generating millions of copies. It requires a repetitive series of the three fundamental steps (one PCR cycle): 1. Denaturation of the DNA template (94-95˚C). 2. Annealing of the primers to the single stranded template (Depending on the primer sequence). 3. Extension (72˚C). APBI 305 Polymerase Chain Reaction APBI 305 Polymerase Chain Reaction Applications: Ø Detection of genetic and infectious diseases Ø Detection of infectious diseases Ø Detection of mutations Ø Forensic applications Ø Generating DNA probes Ø Analysis of ancient DNA Ø Studying gene expression APBI 305 Recombinant DNA Technology It is the use of laboratory techniques to recombine different DNA segments together. It enables researchers to isolate genes and insert them into host organisms. Tools: Ø Donor DNA Ø Vectors Ø Host organism Ø Enzymes e.g., restriction enzymes, ligases, etc. APBI 305 Restriction Enzymes Enzymes that cleave the sugar phosphate backbone of DNA at a specific nucleotide sequence known as the enzyme recognition site. They are normally found in bacteria and archae where they provide a defense mechanism against foreign DNA (e.g. Viruses). A recognition site is a short sequence varying between 4 and 8 bp. The sequence is usually palindromic (the sequence is read the same forwards and backwards on the same DNA strand or on 2 complementary strands in a dsDNA molecule). e.g. 5’..GTAATG..3’ (same strand) 5’..GTATAC..3’ 3’..CATATG..5’ (2 strands, inverted repeat) APBI 305 Restriction Enzymes Restriction digestion results in double stranded DNA with blunt or sticky ends. EcoRI produces "sticky" ends. SmaI produces "blunt" ends. APBI 305 Restriction Enzymes Cleaved DNA fragments could re-form phosphodiester bonds using the DNA ligase enzyme. This is the principle of ligating DNA fragments together in cloning experiments. APBI 305 Plasmid Small extrachromosomal circular dsDNA molecules found in bacterial cells. Plasmids replicate independently. They can be used as cloning and expression vectors to transfer DNA among different species. APBI 305 Cloning Vectors Used to transport and amplify a specific sequence into a host species. Cloning vectors generally contain: Ø Origin of replication Ø A selectable marker to allow identifying recombinants. Ø Multiple cloning site to facilitate insertion of foreign DNA. APBI 305 Expression Vectors In addition to cloning, they are used to express a gene of interest into a protein, e.g., production of human insulin. The plasmid vector is engineered to contain enhancers and promoters to allow gene expression. Gene expression could be constitutively stimulated or induced with an inducer. Cloning eukaryotic genes in prokaryotic hosts: RNA should be reverse transcribed into cDNA (carrying a 5’ cap, 3’ polyA tail and no introns). APBI 305  Tag allowing tagging of Promoter your expressed protein for purification purposes List of restriction enzymes that cut within the vector MCS and from which we choose enzymes to cut our insert. Thrombin cut site allowing cutting your expressed protein from the upstream tag Selectable marker allowing survival of transformant cells Origin of replication APBI 305 Recombinant DNA Technology General Procedure: 1. DNA is extracted from the host organism or in vitro generated as cDNA from RNA transcripts. 2. Insert is cloned into vector by 3 main strategies (depending on your aim and design): a) Sticky end ligation Insert and vector are digested with restriction enzymes that generate sticky complementary ends. Adapters or ligators can be added to your insert during PCR amplification to facilitate cloning. APBI 305 Recombinant DNA Technology b) Blunt end ligation Insert and vector do not require restriction digestion/digestion with blunt end enzymes e.g. EcoRV Blunt-ended vectors are usually de-phosphorylated to minimize self-ligation. Therefore PCR products (which are usually 5’ dephosphorylated) need to be phophorylated before cloning. To increase efficiency: increase DNA and ligase concentration. APBI 305 Recombinant DNA Technology c) TA cloning No restriction enzymes are used. An adenine is added to the 3’ end of your insert by Taq polymerase during PCR. Your insert complements with vectors carrying a thymidine on the 5’ end. Insert and vector are ligated normally by DNA ligase. PCR Purchased APBI 305 Recombinant DNA Technology 3. Recombinant vector is transformed into the host cell (e.g. DH5α competent cells). 4. Cells are grown on agar plates overnight at 37°C. 5. Successful transformants and/or recombinants are selected using selectable markers (usually antibiotics) and regrown overnight in flasks at 37°C in a shaking incubator (plamid miniprep). 6. Additional: Cells could be further grown in bigger flasks (plasmid medi and maxiprep). 7. DNA is extracted from bacterial cells. 8. Insert uptake could be further verified. e.g. by PCR, restriction digestion, sequencing. 9. Next steps depend on your aim, such as: In vitro transcription of your gene to be labelled and used as an RNA probe. Expression and purification of a protein to be used as a drug. Packaging of your construct into AAV9 virus to be used in gene therapy. APBI 305 Recombinant DNA Technology APBI 305 Selection and Screening In order to identify successful transformants and recombinants, vectors are engineered to contain selection and screening markers. Common selection markers are genes that confer resistance to antibiotics. e.g. β-lactamase confers resistance to ampicillin. Selection markers allow the identification of transformants (cells bearing the vector). APBI 305 Selection and Screening A common screening strategy: Blue-white color screening that is used to distinguish bacterial cells containing the recombinant vector from those containing the parental vector (not bearing the insert). β-galactosidase enzyme is expressed by LacZ gene which in the presence of IPTG (inducer) and X-GAL (substrate) results in blue colonies. Successful cloning of your gene of interest will lead to the disruption of the LacZ gene and therefore the growth of white colonies. APBI 305 Selection and Screening White Colonies: Bacterial colonies containing the recombinant vector APBI 305 Selection and Screening APBI 305 Cloning Applications Genome sequencing Production of recombinant proteins Generation of probes Gene therapy Transgenic organisms APBI 305  Exercise on Lecture 1 You would like to clone and express the human RACK1 gene in the pAB-6xHis-MBP™ expression vector. Please answer the following questions: A. Using NCBI, what will be the sequence you are going to clone? B. What are the basic steps of your cloning experiment? C. How can you screen for successful recombinants? D. What will be the size of your recombinant vector? E. What will be the size of your expressed protein (in amino acids)? F. Using the vector elements, how can you purify your recombinant protein? APBI 305 APBI 305 THANK YOU