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Gene Cloning

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Gene Cloning PLNT2530 2021 Unit 6a Gene Cloning Vectors Molecular Biotechnology (Ch 4) Principles of Gene Manipulation (Ch 3 & 4) Analysis of Genes and Genomes (Ch 5) Unless otherwise cited or referenced, all content of this presenataion is licensed under the Creative Commons License 1 Attribution Share-Alike 2.5 Canada Plasmids Gene 1 Naturally occurring plasmids ori -occur widely in bacteria -are covalently closed circular dsDNA -are replicons, stably inherited as extra-chromosomal DNA -can be 1 kbp to 500 kbp in size (compared to 4000 kbp chromosome) -bacteria can contain several different types of plasmid simultaneously -many naturally occurring plasmids carry genes for restriction enzymes, antibiotic resistance, or other genes 2 Bacterial Vectors All vectors : 1. -must replicate autonomously in ori - origin of replication their specific host even when sequence at which DNA polymerase joined to foreign DNA initiates replication 2. - should be easily separated from host chromosomal DNA E. coli chromosomal DNA: ~ 4 million bp typical plasmid vector: ~ 3 to 10 kb Most modern cloning vectors in E. coli are derived from naturally-ocurring plasmid col E1. Most of col E1 was deleted except for an origin of replication and an antibiotic resistance gene. 3 Vectors Types cloning small plasmids- can occur naturally in as circular dsDNA in fragments bacteria (up to 15 kb) eg. single genes bacteriophage -viruses of bacteria (~10-50 kb) used in the cDNA cloning, high-efficiency construction of cDNA and genomic libraries cloning BAC-bacterial artificial chromosome (130-150 kb genomic libraries YAC-Yeast artificial chromosome (1000-2000 kb) with large inserts Each type of vector has specific applications but primary function is to carry foreign DNA (foreign to bacteria) and have it replicated by the bacteria 4 Introduction of foreign DNA into E. coli “Competent” bacterial cells Bacterial cells with plasmid Plasmid vector transform Lyse cells and recover plasmids Plasmid transformed bacteria Recombinant Isolate plasmid vectors plasmid vectors Cut with a restriction enzyme to linearize without fragmenting Add foreign DNA with compatible ends and ligate 5 Plasmids ori Plasmid vectors (engineered) should 1. have naturally-ocurring copy number control sequences deleted Result: higher plasmid copy number in the bacteria (eg. 20-200 copies/cell) eg. pUC19 2. be small in size (3-5 kbp) ● to facilitate separation from chromosomal DNA ● transfer efficiency declines for plasmids >15 kbp 3. carry selectable markers to allow selection of: i) plasmid transformed bacteria ii) transformed bacteria that carry the recombinant vector 6 3. Selectable markers -explanation i) selection of plasmid-transformed bacteria Achieved by engineering a plasmid to carry and express a gene for antibiotic detoxification which will allow any bacterium carrying that plasmid (and gene) to be resistant to the toxic effects of the antibiotic. Following transformation of bacterial cells (antibiotic sensitive without this plasmid), cells are plated on an antibiotic containing media. Commonly used antibiotics: Ampicillin, streptomycin, kanamycin, hygromycin Removes non-transformed bacteria which will be present in the transformation mixture 7 Bacterial transformation Competent bacterial Plasmid with cells sensitive to antibiotic resistance antibiotic cmpd gene Bacterial colony derived from a single cell A significant % of cells are not transformed Petri plate with bacterial agar media containing an appropriate antibiotic 8 3. Selectable markers Antibiotic selection - kills all cells that did not get the vector However - there is no guarantee that all vectors got an insert. Therefore, many cloning vectors let us screen for those with inserts based on insertional inactivation of a gene. Example: X-gal assay for the LacZ gene 9 Expression of LacZ gene (5-bromo 4-chloro 3-indolyl β-galactoside) this is what happens if we don’t insert a fragment X-gal Ori (colourless substrate) Plasmid Expression of gene β-galactosidase in bacterial cell r galactose Amp LacZ gene Released indolyl cmpd dimerizes after oxidation to yield an insoluble indigo dye (blue color) bacterium Colonies expressing LacZ gene NOTE: X-gal is taken up by cells but the product becomes insoluble inside the cells Agar plate Amp X-gal 10 Selection for bacteria carrying recombinant plasmids Ori Plasmid Restriction enzyme Foreign DNA LacZ gene Ampr Ampr DNA Ligase Amp + X-gal Plasmid Will die in presence of Non-recombinant antibiotic Transform plasmid bacteria Will grow and produce Plate on media blue colonies with antibiotic Recombinant and X-gal plasmid Will grow and produce 11 White colonies • X-Gal structure 12 Plasmids Plasmid vectors (engineered) should: (cont’d) 4. Have a multiple cloning site (MCS) region A constructed ds sequence containing a series of consecutive, unique (ie only one site in the plasmid) restriction enzyme recognition sites. The sequence is a multiple of 3 bp long and is inserted into the coding region of the selectable marker (i.e. LacZ gene) gene. Sequence is relatively short (54 bp in pUC19) As multiple of 3, the insertion does not change the reading frame of coding region of the gene, adds 18 amino acids to protein - but doesn’t affect product activity. Value: Allows different restriction enzymes to be used to open plasmid to match the restriction enzyme used to create the DNA being inserted. 13 Multiple cloning site (MCS) from pUC 18/19 plasmids EcoRI SacI KpnI BamHI XbaI gaattCGAGCTCGGTACCCGGGGATCCTCTAGA SmaI SalI PstI SphI HindIII 10 unique restriction enzyme sites GTCGACCTGCAGGCATGCAAGCTTGGctg EcoRI HindIII P pUC18 HindIII EcoRI P pUC19 Paired vectors have oppositely oriented MCS 14 In-frame insertion of MCS adds 12-18 amino acids EcoRI MCS HindIII P pUC18 pUC18 Transcription into mRNA Translation of mRNA into protein End is generally away from catalytic site 15 Example: Insertion at an Eco RI site EcoRI K B X P HindIII EcoRI site LacZ - MCS E E Insertion of foreign DNA EcoRI ends into vector 16 Example: Insertion at a Bam HI site EcoRI K B X P HindIII B LacZ - MCS B B 17 Fragments can be inserted using either 1 or 2 restriction sites EcoRI K B X P HindIII B B K B LacZ - MCS E E E P K K K X Etc, etc X X X H P P E P H H E H 18 Example: One enzyme, two possible orientations E EcoRI K KpnI H S Plasmid X XbaI E K EX H S SalI S MCS X K H HindIII E Promoter Possibilities? Goal: Express Red gene by Green promoter in plasmid 19 Example: One enzyme, two possible orientations E EcoRI K KpnI H S Plasmid X XbaI E K EX H S SalI S X Cut with E K H HindIII Isolate red gene E Promoter Cut with E H E S X K E E E Ligase Goal: Express Red gene by Green promoter in plasmid 20 Example: One enzyme, two possible orientations E EcoRI K KpnI H S Plasmid X XbaI E K EX H S SalI S X Cut with E K H HindIII Isolate red gene E H S Promoter XK Cut with E E H E S X K E E E E H S XK Ligase E E Goal: Express Red gene by Green promoter in plasmid 21 E When the same restriction enzyme cuts on both ends, the insert can be ligated to the vector in either of two possible orientations. GAATTC vector vector CTTAAG 5’ 3’ G AATTC vector G AATTC vector CTTAA G CTTAA G 180° 5’ 3’ vector G AATTC G AATTC vector CTTAA G CTTAA G 22 Example: Two enzymes, one possible orientation This is called directional cloning Orientation is defined with reference to the direction of transcription H S H S E X K E E X E K 23 Example: Two enzymes, one possible orientation This is called directional cloning E EcoRI K KpnI H S Plasmid S SalI E K EX H S H HindIII X K X XbaI E Promoter Goal: Express Red gene by Green promoter in plasmid 24 Example: Two enzymes, one possible orientation This is called directional cloning E EcoRI K KpnI H S Plasmid S SalI E K EX H S H HindIII X Cut with K and X K Isolate red gene X XbaI E Promoter Cut with K and X H X S X K K E Ligase Goal: Express Red gene by Green promoter in plasmid 25 Example: Two enzymes, one possible orientation This is called directional cloning E EcoRI K KpnI H S Plasmid S SalI E K EX H S H HindIII X Cut with K and X K Isolate red gene X XbaI E Promoter Cut with K and X H X S X K K E X Ligase K Goal: Express Red gene by Green promoter in plasmid 26 Bacteriophage Derived Vectors – 2nd type of vector Common phage (virus) of E. coli is lambda Viruses typically consist of a DNA (RNA) based genome and a protein coat A virus uses host cells to replicate and express their genome Features of natural lambda ds DNA , ~49 kbp, encodes 67 genes outside its host, phage exists as a phage particle (capsid particle) linear dsDNA wrapped in a protein coat ds except for both 5’ termini which are 12 nt extensions the extensions are complementary allowing circularization The 12 nt extension is called a COS site 27 Bacteriophage Lambda Complementary 12 nt COS sequence ~49 kb Head dsDNA COS sequence allows circularization Tail of the genome inside a host cell Capsid particle 28 Bacteriophage Lambda from http://www.bioinformatics.nl/molbi/SCLResources/lambda.htm 29 Bacteriophage lambda (λ) infection of E. coli Bacterial cell Host chromosome Adsorption to host cell and insertion of phage DNA λ DNA Circularization of λ DNA Integration of λ DNA Expression of λ genes into host chromosome Prophage 30 Lysogenic pathway Infection (cont’d) Integration of λ DNA Expression of λ genes into host chromosome • Replication of λ DNA Induction event • Synthesis of head and tail proteins • Packaging in particles Lysogenic pathway • Host cell lysis • Release of infectious particles Lytic pathway 31 Lambda genome DNA replication Head Tail Lysis 0 10 20 30 40 kbp Only one gene is expressed in the lysogenic pathway - cI repressor protein which prevent the first stage in the sequential expression of the lambda genome genes.
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