Biotechnology Basics Lecture Slides PDF - Mark Carlile

Biotechnology: The basics Mark Carlile Dale 1.08 [email protected] Biotechnology Intro | Mark Carlile 1 Overview: This week we will cover: The basics of biotechnology – the aims A little historical perspectives A look at the current (and future)...

Biotechnology: The basics Mark Carlile Dale 1.08 [email protected] Biotechnology Intro | Mark Carlile 1 Overview: This week we will cover: The basics of biotechnology – the aims A little historical perspectives A look at the current (and future) trends in biotechnological research Biotechnology Intro | Mark Carlile 2 What is biotechnology The use of biological systems to generate useful products More specifically: It is the exploitation of biological processes for industrial and other purposes, especially the genetic manipulation of microorganisms for the production of antibiotics, hormones and recombinant At its core Biopharm Science is proteins Biotechnology Biotechnology Intro | Mark Carlile 3 Holistic aims of modern biotechnology Biotechnology Intro | Mark Carlile 4 Historical biotechnology Biotechnology is probably ~ 6000 years old: 6000 BC : Egyptians used yeast to leaven bread and make alcohol via fermentation 1521 : Aztecs cultivated and harvested algae as a food source 1869 : Johan Meischer isolates DNA from WBCs 1928 : Alexander Fleming : Discovery of penicillin from a bread mold 1953 : Watson and Crick : DNA structure elucidation 1973 : Genentech produce human insulin in E.coli Biotechnology Intro | Mark Carlile 5 Future focus of biotechnology Biotechnology is now embracing all of the current recombinant DNA and cellular engineering technologies Examples: Transgenic animals Gene editing Multigene cassettes – multiple products/synthetic pathways (Biotransformation) Tissue regeneration Protein production and protein engineering Gene therapy Biotechnology Intro | Mark Carlile 6 Exogenous gene expression Expression of non-native genes in an organism Expression is driven via host cell expression “machinery” Use of native and non-native promoters Expression can be via plasmids or via integration into the host genome Biotechnology Intro | Mark Carlile 7  Expression vectors F1 phage origin : viral packaging Reporter gene Antibiotic resistance Protein tag – assay/purification Origin or replication Promoter (T7 phage) lacI gene Various restriction enzyme sites are present Biotechnology Intro | Mark Carlile 8  Nucleic acids: Manipulation: Cutting Restriction enzymes DNA can be cut in a sequence-specific manner by restriction endonucleases. These enzymes bind to specific DNA sequences and break phosphodiester bond in both strands to generate sticky or blunt ends Compatible sticky ends will anneal because of complementary base pairing Frequency in cutting dependent on the length of recognition sequence: 4 bp recognition seq cut ~ every 256 bp 6 bp recognition seq cut ~ every 4096 bp 8 bp recognition seq cut ~ every 65,536 bp Dr Mark Carlile | DNA/RNA Protein in the 9 Lab  Cloning into an expression vector When cloning any gene : moving it from one DNA sequence to another – you need to have restriction sites flanking (at the ends of) the gene YGOI = Your gene of interest Orientation is important Cut both DNA molecules: donor and receiver with the same restriction enzymes usually use different Res-Enzyme to stop re-ligation and to preserve orientation of the insert Gel purify the two fragments : Gel electrophoresis – band extraction – DNA purification Join the fragments together using DNA Ligase (T4 DNA ligase) usually use 3X as much insert as receiver vector : e.g 3 ug insert : 1 ug receiver plasmid Verify cloning via gel electrophoresis Biotechnology Intro | Mark Carlile 10 Expression vectors: Eukaryotes Eukaryotic expression vectors are constructed and amplified (get more DNA) in microbial systems The basic components are the same Most use viral promoters : CMV/SV40 for strong expression More specific promoters will be used for lower expression levels (control) or for cell-specific expression patterns (mammalian expression) Biotechnology Intro | Mark Carlile 11  Expression systems Expression Host Your expression system will generate your protein product Mammalian Cells Bacterial / Yeast Cells You have to choose your expression format at the start of your cloning work. Correctly folded and fully Intracellular post-transcriptionally modified Expression Soluble Insoluble Transported to the Culture media Available for purification Extracellular Soluble Cell Breakage Folded (PT Modified?) Required Expression Available for Purification and modification (if required) Dr Mark Carlile : BPS302 Week 15 (In-class 12 slides) Expression vectors and the E.coli lac operon Dr Mark Carlile : Biologics Slide 13 Recombinant vectors Controllable Promoter (inducible) Origin of replication Transcriptional control elements (ori) Ribosome binding site (ATG) pBPS302 Targeting Sequence (N-term) plasmid Gene of interest (CDS) Selection gene (ampr) Targeting Sequence (C-term) Termination sequence Expression cassette lac lac lac PlacI lacI Plac Olac A Z Y MKKTAIAIAVALAGFATVAQA (ompA Signal Sequence) Example: the lac operon Dr Mark Carlile : Biologics Slide 14 The lac operon lacZYA transcription cassette Polycistronic mRNA lac lac lac PlacI lacI Plac Olac Z Y A lacZ : β-galactrosidase Wild-type molecular switch lacY : galactoside permease lacA : thiogalactoside transacetylase lac repressor (Homotetramer) lac Operator Plac Wild-type molecular switch 28 bp 28 bp Binging of lacI to the operator is RNA Poly’ase + binding cooperative Plac Binding of lacI to Olac promotes the binding of RNA polymerase Low level expression of lacZYA lac lac lac A Y Z Dr Mark Carlile : Biologics Slide 15 The lac operon RNA Poly’ase + binding Plac Wild-type molecular switch Un-induced Binging of lacI to the operator is lac lac lac A Y Z cooperative Binding of lacI to Olac promotes the binding of RNA polymerase Low level expression of lacZYA + RNA Poly’ase β-galactosidase Plac Induced Galactoside permease (lacY) allows lactose lac lac lac uptake from media A Y Z β-galactosidase (lacZ) converts lactose to: - Glucose + Galactose (main reaction) - Allolactose (side reaction) Allolactose binds to lacI and changes its conformation RNA Poly’ase lacI repressor tetramer dissociates from Plac the operator (Olac) Transcription of lacZYA genes Induced RNA Poly’ase lac lac lac A Y Z Transcription Dr Mark Carlile : Biologics Slide 16 Use of the lac operon control elements for heterologous protein expression Binging of lacI to the operator is RNA + Poly’ase binding cooperative Plac Binding of lacI to Olac promotes the binding of RNA polymerase Low level expression of lacZYA lac lac lac A Y Z Gene of lac lac lac Interest A Y Z Molecular Biology Molecular Biology (Cloning-in) (Restriction enzyme cleavage) RNA + Remove the lacZYA genes – restriction cleavage binding Clone-in you gene of interest Poly’ase Plac Control by IPTG induction Gene of Interest This promoter-expression-cassette works but is prone to leakage (switched on in the absence of lactose) Need a more tightly controlled promoter-expression-cassette Leaky expression leads to plasmid instability – plasmid loss Dr Mark Carlile : Biologics Slide 17  Expression vectors: Chinese hamster ovary cell and mammalian expression systems Dr Mark Carlile : Biologics Slide 18 Mammalian expression system Production of recombinant proteins in mammalian cells has allowed the manufacture of a number of large, complex glycosylated polypeptides for clinical applications The mammalian host cell of choice is the Chinese Hamster Overy (CHO) cell CHO cells are adherent but can be grown in stirred bioreactors as a suspension (Protein Expression) The cells require proline in the medium for growth. CHO cells are maintained suspended in culture by revolving culture continuously at approximately 50 RPM. CHO cells should be cultured in DMEM modified with 10% FBS. If cells are not doubling every 14-17 hours, supplement the medium with 1-2% FCS. DMEM - Dulbecco's Modified Eagle Medium FBS – Fetal bovine serum FCS – Fetal calf-serum Can take a while to generate a stable line (expressing) Once the cell line is growing Dr Mark Carlile : Biologics Slide 19 Mammalian expression system: The CMV Promoter An example mammalian expression promoter: The strong human cytomegalovirus (hCMV) promoter regulatory region drives constitutive protein expression levels as high as 50 mg/L. For less potent cell lines, protein levels are typically ~0.1 mg/L. The presence of the SV40 replication origin will result in high levels of DNA replication in SV40 replication permissive cells. CMV vectors contain the pMB1 (derivative of pBR322) origin for replication in bacterial cells, the β-lactamase gene for ampicillin resistance selection in bacteria, hGH polyA, and the f1 origin. Vectors containing the pre-pro-trypsin leader (PPT) sequence direct secretion of fusion proteins into the culture medium for purification using antibodies, resins, and plates. The CMV Promoter: cis acting sites Species CDS Enhancer Seq Promoter Leader Seq Unique Seq -550 -50 +1 +100 Dr Mark Carlile : Biologics Slide 20 Mammalian expression system: The CMV Promoter Cellular Differentiation Heat shock Serum Stress Ca++ cAMP TNF-α PKC PKA IE1 CREB: cAMP Response element binding ELK- SRF 1 CRE CCATATATGG--TTCCG NF- B SRE ETS κB CCATTGACGTCAATGG GGGACTTTCC CRE NF-κB Unique Seq Enhancer Seq Promoter Leader Seq CDS Complex regulatory pathways CMV is a herpes virus that infects most cell types and establishes latency in leukocytes. The control of the CMV promoter involves many interconnected cellular pathways that both up- and down- regulate expression The enhancer sequence is the primary control point Industrial users of this promoter have modified the enhancer sequence and its regulation to their own benefit - Proprietary /Patents Dr Mark Carlile : Biologics Slide 21 Expression host overview Biotechnology Intro | Mark Carlile 22 Making a COVID-19 Vaccine Restriction enzymes SARS-CoV Spike Protein gene SARS-CoV Spike Protein gene Ligase enzymes SARS-CoV Spike Protein gene Transformation SARS-CoV Spike Protein gene Selection of clones Biotechnology Intro | Mark Carlile 23 Making a COVID-19 Vaccine Cell breakage (Homogenisation) Shake flask Cell Harvest Production fermenter (Centrifugation) x3 Final product Formulation Chromatographic separation Biotechnology Intro | Mark Carlile 24 Summary: This week we have covered: The very basics of biotechnology: what it is and what it does We have looked at basic cloning protocols Looked at the different expression systems that are commonly used in biotechnology/bioprocessing Biotechnology Intro | Mark Carlile 25