Lecture 15: Basics of Recombinant DNA - PDF

Basics of Recombinant DNA Technology 4BICH001W Biochemistry Dr Sarah K Coleman Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous).  Le...

Basics of Recombinant DNA Technology 4BICH001W Biochemistry Dr Sarah K Coleman Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous).  Learning Outcomes Have an overview of the procedure of DNA recombination and gene cloning Be able to describe the role of restriction endonucleases and ligases Know the basic requirements for a plasmid vector Be able to list some of the properties of a host cell for recombinant DNA technology Be able to give examples of applications using the technology  What is Recombinant DNA? Recombinant DNA is segments of DNA from two or more sources (often different species) linked together e.g. jelly fish, human and bacterial DNA Usually DNA sequences that would not normally occur together They are combined in vitro into the same DNA molecule Paul Berg won Nobel Prize for Chemistry 1980 for “his fundamental studies of the biochemistry of nucleic acids, with particular regard for recombinant DNA.” https://www.nobelprize.org/prizes/chemistry/1980/berg/facts/ DNA (Gene) Cloning It is normally cDNA used (not the entire gene). cDNA is made from mRNA as outlined in previous lecture. Aim is to amplify and make many copies (but not by PCR). Then DNA or its encoded protein is used for either basic research or in bio-technology application Applications of DNA Cloning Basic biosciences research Proteins structures; creation of model systems Medicine Gene therapy; vaccines Agriculture Plant modification; farm livestock (mammals); fish Bio-tech industry Pharmaceuticals; antibodies; enzymes; other proteins; other industrial chemicals  DNA Cloning: A preview Most methods for cloning pieces of DNA in the laboratory share general features e.g. use of bacteria and their plasmids and specific enzymes Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome Cloned DNA is useful for making copies of a particular gene and/or producing a protein product. Gene cloning involves using a host cell (e.g. bacteria) to make multiple copies of a gene DNA Cloning: A preview Foreign DNA is inserted into a plasmid and the recombinant plasmid is inserted into a bacterial cell Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA This results in the production of multiple copies of a single gene (or piece of DNA) Recombinant plasmids are first step in creating tools for more complex GMO manipulation  The Process Overview 1 Figure 9-1 part 1 Lehninger Principles of Biochemistry, Fifth Edition © 2008 W. H. Freeman and Company  The Process Overview 2 Figure 9-1 part 2 Lehninger Principles of Biochemistry, Fifth Edition © 2008 W. H. Freeman and Company The Plasmid Also known as a vector Originally small independent bacterial DNA which could be shared and transmitted (antibiotic resistance) Replicate independently of bacterial chromosome (have own origin of replication) Nowadays highly engineered and modified for particular research purposes e.g. https://www.addgene.org/vector-database/ MUST have some fundamental properties / aspects for research use  Example of a plasmid Antibiotic resistance gene for A Multi-Cloning Site selection (MCS) where new fragment of DNA will be inserted. Lots of unique restriction enzyme sites Origin of replication so plasmid can replicate in bacteria  The Recombinant Plasmid Complimentary ends e.g. ATA ATAGCGCTT TATTATCG CGAA DNA cut with Restriction Endonucleases DNA joined together by complementary ends and T4 Ligase  Recombinant plasmid inserted into host bacterium by transformation process This is gene cloning NOT reproductive cloning And you have now made a GMO Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous).  Some Essential Terminology a) Sequence databases and use of programs to analyse them. A. Bio-informatics b) Complementary DNA. Produced from mRNA. B. cDNA c) Isolating and amplifying a gene in a plasmid; usually only the C. Gene Cloning protein encoding sequence via cDNA. D. Gene Editing d) Specifically modifying a gene within an organism's genome. E. Genetically e) (GMO) an organism which has had its genetic make-up changed. Could be a bacterium, could be a plant or animal… Modified Organism f) Bacterium a recombinant plasmid is inserted into for F. Host Cell amplification G. Ligases g) Enzymes which join DNA strands together. H. Plasmid (vectors) h) Small, circular, extra-chromosomal DNA. With origin of I. Recombinant DNA replication. J. Restriction i) DNA from different species combined in vitro. j) (RE) Enzymes which cut DNA. Ones with defined and specific Endonucleases recognition sequences are used.  Key Enzymes: Restriction Endonucleases (REs) Originally isolated from bacteria (part of defence system) Now commercial ones are produced via biotechnology and have been modified Cut within a double stranded DNA sequence Three main types: Type I cut at random far (~1000 bp ) from the recognition sequence Type II cut at defined position - within a specific recognition site Type III cut ~25bp from recognition site and requires ATP Only Type II are used in DNA analysis and gene cloning Recognition sites are known and can be predicted in a given DNA sequence  Some REs in plasmid MCS with recognition sequences Part of recombinant plasmid Beginning of encoded protein Restriction enzyme map of cDNA encoding Stargazin protein http://nc2.neb.com/NEBcutter2/  Restriction Endonucleases (REs) Key feature is uniqueness and specificity of an enzyme for a particular recognition site http://rebase.neb.com/rebase/rebase.html Naming of REs has no relation to recognition site, comes from bacteria first isolated from. e.g. EcoRI from Escherichia coli (strain R, 1st isolated) e.g. PvuII from Proteus vulgaris (2nd isolated) REs can produce ‘sticky’ ends or ‘blunt’ ends Sticky ends preferred for cloning… REs are bacterial defence against bacteriophages Often occur with DNA-methyl transferases Recognise same sequence and add -CH3 Restriction-Modification Systems methyl groups prevent the restriction enzyme from cutting the DNA protects bacterial DNA a restriction enzyme and its "cognate" modification enzyme(s) form a restriction- modification (R-M) system.  Key Enzymes: DNA Ligases Enzymes which repair single-stranded breaks in double stranded DNA (so replacing a phosphodiester bond) T4 DNA ligase is used in vitro for gene cloning Originally from bacteriophage; now modified and produced by biotechnology. RE producing sticky ends preferred for cloning…why? Blunt End Ligation vs Ligating Sticky Ends more efficient  Process of DNA Cloning Lehninger Principles of Biochemistry, Fifth Edition © 2008 W.H.Freeman and Co Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Discussion Board in the Biochemistry Blackboard module and I will look at them later (asynchronous)  Inserting Recombinant Vector into Host Cells Transformation of Bacteria Cells prepared beforehand Easy to manipulate Rapid growth Easy to select for Heat shock Remember: Plasmids have an ANTIBIOTIC resistance gene This allows selection of transformed bacteria  Bacterial Host Cells Types of Escherichia coli (E. coli) Gram negative bacterium Very well studied Many different cultivated laboratory strains – all highly modified Some strains produce very high quality and pure recombinant plasmid DNA Some strains engineered to efficiently produce recombinant proteins of interest http://blog.addgene.org/plasmids-101-common-lab-e-coli-strains Next steps…other host cells or organisms?  Bacteria as Model Organisms Pro’s Con’s Cheap No RNA splicing machinery Easy to grow and fast No protein modification Easy to manipulate machinery (e.g. glycosylation) Easy to screen / select for Not the eukaryote cell / organism of interest Lots of modified strains available Often using bacteria just to produce many copies (clones) of the desired recombinant DNA. Recombinant vector is ultimately a tool to then manipulate eukaryotic cell lines or organisms.  Plasmid amplified by DNA amplified by PCR growing bacterial cultures and DNA isolation RE digestion RE digestion Ligation Now have recombinant DNA transformation Grow bacteria to make Isolate recombinant plasmid many more copies from bacteria  Your recombinant plasmid can now be used in: Applications of Recombinant DNA Technology Figure taken from Hsu et al (2014) Cell, 157(6), 1262-1278 Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous).  Animal Models Gene knockout: Disrupt a gene in the animal and then look at what functions are affected Determine the role and function of the gene Gene knock-in: Replace normal gene with a mutant version Determine effects of mutant gene function in animal Since humans are similar to rats and mice, these gene studies in rats and mice can lead to better understanding of gene function in humans  Plant Engineering Herbicide resistance Plant made resistant to herbicide, use newer more environmentally friendly herbicides e.g. Round Up Soya Plant vaccines Plants produce own pesticide – reduces spraying by farmer e.g. Bt cotton Enhanced nutrition Plant expresses beneficial nutrient e.g. Golden rice produces beta- carotene, a precursor to Vitamin A Industrial Protein Production Examples of products from recombinant DNA technology: Tissue plasminogen activator, to prevent and dissolve blood clots Human growth hormone, to treat pituitary dwarfism. Human blood clotting factor VIII, to treat haemophilia. Human insulin (“humulin”) for insulin-dependent diabetes. Recombinant vaccines Monoclonal antibodies Genetically engineered bacteria specialized to degrade pollutants  CRISPR /Cas9 and genome editing Made eukaryotic genome editing easier and cheaper; was not first method! Allows specific genes (or parts of) to be removed or replaced Done in vivo in organism High precision (but not perfect) Was a prokaryotic defence against viruses Emmanuelle Charpentier and Jennifer Doudna Nobel Prize in Chemistry 2020 https://www.nobelprize.org/prizes/chemistry/2020/summary/  CRISPR / Cas9 and genome editing CRISPR sequences (crRNA) are designed by experimenter. To guide complex to correct genome location. Cas9 is the enzyme acting as ‘scissors’. Not specific for cut site. Other component are tracrRNA (for active complex creation). Can combine all required RNA sequences and Cas9 cDNA into a plasmid to then transfect / infect into eukaryotic cells. Can also include a ‘DNA repair template’ (DNA to be inserted) DNA cut on both strands MUST be repaired by the cell Native repair mechanism utilised. However…  Repair Mechanisms used in CRISPR/Cas9 Can KNOCK OUT gene function by triggering Non-Homologous End Joining Extra nucleotides get inserted, causing a mutation Stops gene expression and function Can KNOCK IN a new gene by Homology Directed Repair This uses provided DNA repair template containing DNA sequence of interested with correctly matched (homologous) ends Cell will repair by inserting new DNA sequence into break. Hsu et al (2014). Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157(6), 1262-1278. (review article)  Summary The goal of recombinant DNA work is to produce many copies clones of a particular gene (or it’s cDNA). The DNA must be introduced into a host cell using a vector A vector is cut using restriction enzymes and DNA of interest inserted using DNA ligase Size of fragments visualised by gel electrophoresis The cells that get the recombinant DNA are distinguished from those that do not by means of genetic markers, called reporter genes e.g. antibiotic resistance Summary Recombinant DNA and its manipulation is a fundamental part of bioscience research Engineered E. coli strains and plasmids (vectors) are used Genetic engineering technology is rapidly evolving - new methods and applications being developed Applied across all domains of life Ethical and safety issues must be considered before developing and using genetic engineering technology Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous). MCQ quiz for Lecture 15: Basics of recombinant DNA Answers will be given in your Seminar sessions – with further discussion. You must attempt before your seminar session. These quizzes are part of your learning for the Biochemistry module They will aid your on-going studies at the University of Westminster Q1) Recombinant DNA is used in research because it ____? a) Enables tagging and tracking of proteins in cells, so assisting in research on protein function. b) Enables multiple copies of cDNA to be easily and quickly produced. c) Enables the production of reagents for further gene editing applications. d) Enables scientists to produce fluorescent organisms. e) All of the above. f) Some of the above. Q2) Which of the following statements about plasmids is incorrect? a) For use in research all plasmids must have a bacterial origin of replication, a multi-cloning site and a reporter gene. b) Plasmids used for research nowadays are highly modified from the naturally occurring and original bacterial versions. c) Plasmids are circular pieces of DNA which can be replicated in bacteria. d) Plasmids are always replicated (many copies made of) by PCR. e) Plasmids for research always contain an antibiotic resistance gene. Q3) Enzymes are vital tools in recombinant DNA work. Select the incorrect statements below. Why are they incorrect? a) Ligase enzymes joins fragments of DNA together. b) Type II restriction endonucleases have a specific recognition site (combination of bases) in DNA molecules. c) Methyl transferase enzymes do not need to be considered in planning a DNA cloning strategy. d) Polymerase enzymes are used to amplify DNA in PCR procedure. e) The enzymes used in molecular biology are mainly derived from eukaryotic organisms.  Q4) Correct terminology is important for accurate science communication. Match the terms (a-e) and best available definition (1-8). 1) Combining DNA from distinct species a) Bioinformatics 2) Producing a DNA sequence b) Transformation 3) Inserting cDNA into a bacterium c) Gene cloning 4) Inserting a vector into a bacterium d) Recombinant DNA 5) Joining together of two pieces of DNA e) Gene editing 6) Use of computers to analyse DNA and protein sequences 7) Making a targeted change to an organisms genome 8) Making copies of cDNA via a plasmid and host cell Q5) Recombinant DNA manipulation is an essential application for bioscience research. Which of the following statements are correct. a) Model organisms are a key resource and include both prokaryotes and eukaryotes. b) Ethics and safety must always be considered and planned at the first stages. c) Creating a recombinant plasmid is the first step in genetically modifying a more complex organism. d) All of the above statements are correct. e) None of the above statements are correct.