Construction of Recombinant Microbe What is Recombinant Microbe A microbe which is genetically modified by applying DNA Recombinant Technology A microbe which acquires foreign gene through DNA Recombinant Technology An organism is called “transgenic” if it has genetic information added to it from different type of organism Recombinant DNA technology A DNA technology that utilizes the power of microbiological selection and screening procedures to allow investigators to isolate a gene that represents as little as 1 part in a million of the genetic material in an organism. Recombinant DNA The combination of fragments of DNA from different sources. (Cutting and pasting DNA fragments together) Cutting DNA using Restriction enzymes Restriction Enzymes Isolated from various bacteria, restriction enzymes recognize short DNA sequences and cut the DNA molecules at those specific sites. (A natural biological function of these enzymes is to protect bacteria by attacking viral and other foreign DNA) Process Restriction endonucleases cut at defined sequences (palindromic) of (usually) 4 or 6 bp. They cut on both strands of DNA. This allows the DNA of interest to be cut at specific locations. Cuts yield either "sticky" ends, or "blunt" ends. Sticky ends When the ends of the restriction fragments are complementary, EcoRI – recognition sequence 5'---G ‘ AATTC---3' 3'---CTTAA ‘G---5' Blunt ends When the restriction endonuclease cleaves in the center of the pseudopalindromic recognition site to generate blunt (or flush) ends. HaeIII GG‘ CC CC ‘GG Pasting DNA Two pieces of DNA cut with the same enzyme, can be pasted together using another enzyme called "DNA ligase". Pasting DNA Complementary ends (sticky ends) H-bond Ligase forms phosphodiester bond to seal strands together. Restriction enzymes generate fragments that facilitate recombination Process for Recombinating DNA Cut ends in recognition sequence Open DNA Recombine with another piece of DNA cut with the same restriction enzyme Use ligase to seal the cuts and rejoin the fragments Experimental Design Plasmid Vectors Ori (origin of replication) Polylinker cloning sites Regulatory region (lac operon) Antibiotic resistance gene(s) Reporter gene for protein – color or fluorescent molecule pGlo (an example of plasmid vector) Ori Polylinker cloning region Amp (beta lactamase for resistance) araC( arabinose operon) pBad promoter Green fluorescent protein - reporter Other desirable properties of Vector High copy number Inducible promoter under stringent control Stable incorporation (especially for improvement of microbial traits) Regulating protein expression in E. coli Expression often deleteriously affects growth of the host cell Therefore, expression is usually tightly regulated using specific promoter constructs Expression is the divided into two main phases: Cell growth phase (biomass generation) - expression switched off Expression phase - expression induced Control of expression using the lcI repressor pET expression in E. coli E. coli expression vector pUC features: ori AmpR multiple cloning sites T7 & SP6 promoters Regulated expression: tac promoter (fusion between trp and lac promoters) regulated like lac (IPTG) Fusion protein: purification tag cleavable with Xa protease Purification of recombinant fusion proteins expressed in E. coli Properties of Host (especially for production of recombinant protein) Rapid growth Cheap substrates Not fastidious Low toxicity/pathogenicity Expression hosts (bacteria) E. coli Very well understood genetics and fermentation, rapid growth, not fastidious, wide range of vector systems, very easy transformation, intracellular protein, low yields Expression hosts (bacteria) Bacillus Very well understood genetics and fermentation, difficult transformation, very rapid growth, not fastidious, intracellular protein, high yields, limited range of vectors Expression hosts (bacteria) Streptomyces Well understood fermentation, difficult transformation, moderate-slow growth, not fastidious, extracellular protein, high yields, limited range of vectors Expression hosts (yeast) Saccharomyces Very well understood fermentation, difficult transformation, fast growth, not fastidious, extracellular protein, high yields, limited range of vectors Expression hosts (fungi) Trichoderma Poorly understood fermentation, difficult transformation, slow growth, not fastidious, extracellular protein, high yields, limited range of vectors How to put plasmid into an E. coli cell? ORI Cells that do not Amp R. take up plasmids ORI Amp R. die on ampicillin plates Plasmid vector Enzymatically Mix E.coli cells with insert DNA into plasmids in presence of + plasmid vector Recombinant plasmid CaCl2 Culture on nutrient Transformed E.coli cell agar plates containing survives DNA fragment ampicillin to be cloned Bacterial chromosome Independent plasmid replication Cell multiplication Transformation Putting a plasmid (a vector of a vector carrying an inserted gene) inside a host cell Transformation (1) PRE-INCUBATION The recipient E. coli cells will be exposed to positively charged calcium chloride (CaCl2) ions. This treatment is meant to stress the bacterium in order to render its cell membrane and cell wall permeable to the donar plasmid. This process will make the recipient E. coli "competent" to uptake the plasmid. Transformation (2) * INCUBATION The plasmid (with amp+ gene) is added to a recipient E. coli suspension, which will now be called E. coli + because it is the one which is being transformed. Another E. coli suspension will act as a control, called E. coli - because it will not be exposed to the plasmid; therefore, it will NOT inherit the gene. Transformation (3) * HEAT SHOCK The recipient cells plus plasmids and the control cells not exposed to the plasmids are briefly exposed to 42 degrees C. This step will maximize the uptake of the plasmid through the wall and membrane of the cells. Colonies of E. coli carrying pGlo E. coli carrying pGlo Recombinant DNA Combination of DNA from organisms from two different sources Bacterial and human Bacterial and plant Viral and human Uses of Recombinant Microbes control Uses of Recombinant Microbes Production of protein for analytical and structural analysis Native and mutant proteins for functional analysis Protein for structural (e.g., x-ray crystallographic) analysis Uses of Recombinant Microbes Production of commercial protein products Industrial enzymes Amylase, amyloglucosidase and xylose isomerase for the starch industry Proteases, cellulases and lipases for the detergents industry Proteases for the cheese industry Penicillin acylase for the pharmaceutical industry Uses of Recombinant Microbes Production of commercial protein products Therapeutic proteins Insulinfor diabetes treatment Interferon-gamma for cancer treatment Factor VIII Erythropoetin Epidermal growth factor Example: Production of recombinant human insulin in E. coli Many diabetic patients need to administer insulin to control their blood glucose levels Lilly Example: Production of recombinant human insulin in E. coli • Insulin is synthesized in pancreatic islet cells. • It is made as a single polypeptide chain = preproinsulin. • Preproinsulin is proteolytically processed to form Insulin • In mature Insulin, the A and B chains are linked by disulphide bridges N B chain A chain C C chain Example: Production of recombinant human insulin in E. coli Synthetic Insulin Chain A and Chain B sequences cloned separately into a lac-based expression vector http://members.tripod.com/diabetics_world/lillys_rdna_insulin.htm Example: Production of recombinant human insulin in E. coli Induce lac expression Mix purified insulin chains A and B Refold under oxidising conditions to promote disulphide bond formation Purification and cleavage of b-gal protein tag from insulin chain Humulin Uses of Recombinant Microbes Improvement of microbial traits Increasing N2 fixation ability Ability to use complex substrate such as, cellulose, xylose, and amylum Resistance to drought, heavy metal & other toxic compounds Integration Vector Plasmid Chromosome legume Fixed nitrogen Fixed carbon (ammonia) (malate, sucrose) rhizobia Biological nitrogen fixation: nitrogenase N2 + 8 flavodoxin- + 8H+ + 16 MgATP2- + 18 H2O 2NH4+ + 2OH- + 8 flavodoxin + 16 MgADP- + 16H2PO4- + H2 1. Rare, extremely energy consuming conversion because of stability of triply bonded N2 2. Produces fixed N which can be directly assimilated into N containing biomolecules Genes involved in N2-fixation Effect of nifH overexpression on nitrogen fixation and plant growth Growth response of P. vulgaris plants (45 dpi) inoculated with R. etli strains in the greenhouse. Images: 1, Noninoculated nonfertilized; 2, inoculated with CFN42 (wt); 3, inoculated with HP55 (nifHcDK); 4, noninoculated fertilized with 10 mM KNO3–2 mMNH4NO3. The use of microbe for plant genetic engineering Agrobacterium tumefaciens and nature’s genetic engineering Nature of the Microbe A. tumefaciens is a Gram-negative, non-sporing, motile, rod-shaped bacterium, Closely related to Rhizobium which forms nitrogen-fixing nodules on clover and other leguminous plants. Possesses a large, natural plasmid called Ti Agrobacterium tumefaciens Attracted to wounds or openings in the plant cell wall Uses acetosyringone to inject into the plant cells Ti plasmid enters the plant cell and integrates randomly into the host Plasmid codes or opines and nopalines two distinctive gene products that lead to tumor production in infected plants Ti plasmid Ti plasmid and genes ori--replication controlled sites tra region--responsible for mobility from bacteria to plant cell vir--induce uncontrolled cell division in the host plant t region (tDNA)--group of genes that control the transfer of the tDNA to the host chromosome Genetic Engineering and Ti Uses of Recombinant Microbes For environmental applications Oil “ eating” microbes – Prince William Sound – Alaska Degradation of mercury in the environment – Clean up of contaminated sites See you ….
Pages to are hidden for
"Recombinant Technology"Please download to view full document