plasmids isolation of plasmid dna from bacteria properties of a cloning/expression vector (plasmid)...
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Plasmids
Isolation of Plasmid DNA from Bacteria
Properties of a Cloning/Expression Vector (Plasmid)
Uses of Expression Vectors
PLASMID DNA ISOLATION FROM E.Coli
Lipopolysaccharide
Peptidoglycan
Plasma MembranePLASMID (3-20 kbp)
Chromosomal DNA (4 Mbp)
Proteins
Proteins
Proteins
Proteins
Proteins
How to isolate plasmid without a commercial kit.
Lipopolysaccharide
Peptidoglycan
Plasma MembranePLASMID (VECTOR) (3-20 kb)
Chromosomal DNA (4 Mbp)
Proteins
Proteins
Proteins
Proteins
Proteins
Sol 1 (EDTA, Glucose, Tris pH8) [Binds Mg2+ (interferes with cell wall integrity), breaks outer layer, inhibits DNAses, maintains osmotic pressure, buffers cells at pH 8]Lysozyme (Digests Peptidoglycan)Sol 2 (SDS, NaOH) (Detergent- lyses the cell, denatures proteins including DNAses)Sol 3 (KAc, Acetic Acid)Phenol/Chloroform extraction (Dissociate proteins from nucleic acids)Proteinase K (proteolytic enzyme to remove remaining proteins. Not denatured by SDS)RNAse (Digests RNA)Precipitation of DNA (Isopropanol rapidly ppts nucleic acids. Ethanol removes remaining salt from preparation)
The Qiagen Miniprep Kit you will be using
Buffer P1- Resuspension Buffer50mM TrisHCl pH 8.010 mM EDTA10 ug/ml RNAse A
Buffer P2- Lysis Buffer200mM NaOH1% SDS
Buffer N3- Neutralizing BufferPotassium Acetate/Acetic Acid
Chromosomal DNA +SDS-protein Complex
Plasmid in suspension
Use of Silica Gel Membrane Technology
Plasmid binds tightly to membrane under high [salt]
Elute Impurities
Plasmid is elutedunder low [salt]
using a commercial kit to isolate your plasmid
How to quantify of your DNA (Plasmid)
1. DNA can be quantified in an agarose gel by comparing the intensity of the fluorescence emitted by an ethidium bromide-stained DNA sample relative to a dilution series of a DNA standard of known concentration.
2. If the sample is pure (free of RNA, protein, phenol, agarose), aSpectrophotometric method based on measuring the amount of UV irradiationthat is absorbed by the bases is accurate and convenient.
-DNA concentration is determined from readings at 260nm.-Values are in optical density units (OD) where 1OD=50µg/ml
-Eg, if OD=0.2, [DNA]= 50µg/mlx0.2=10µg/ml-If DNA has been diluted, then dilution factor must be included
-Eg, (2µlDNA+98µlH2O) with 0.2OD will read:
0.2x50x(1/1000)x50= 500ng/µl
Explain?Can you think of a dilution factor(s) you could use that would allowyou to determine [DNA] without any mathematical calculations?
What is a plasmid ?
Expression VectorShuttle VectorCloning Vector
What are the 3 most important parts of an expression vector ?
1. Origin of replication
2. Selectable marker
3. Cloning sites
Ori
RepliconContains all info necessary to begin and end DNA replicationOrigin of replication (Ori) is a defined location within the replicon where DNA synthesis begins
The ColE1 Replicon
ColE1 naturally occuring plasmid of E.coli.replicates independently of the host genome.
DNA Pol
200-300 bp
RNA I : RNA II INTERACTION
ROP PROTEIN NEGATIVELY REGULATES RNA II BY STABILIZING RNA I: RNA II INTERACTION
Copy number of Vectors
Plasmid Replicon=Origin
Copy number
pBR322 ColE1 15-20
pT7T3.pac ColE1 500-700
Why does pT7T3.pac have a high copy number?
• There is a point mutation in the origin that alters the initiation of RNAI transcription, such that the RNAI:RNAII complex does not form as well as wild-type.
• The region of the origin that encodes Rop protein is deleted.
Why does pT7T3.pac have a high copy number?
2 REASONS
Selectable Marker (Ampicillin Resistance)
Ampicillin: -belongs to family of antibiotics called β-lactams -penicillin derivative
β-lactam ring
Interacts with and inactivates transpeptidase which is responsible for transpeptidation (cross-linking of sugars)
AmpR gene codes for β-lactamase which inactivates ampicillinby cleaving the β-lactam ring.
AmpR gene: to select for bacteria containing plasmid.
-Why does bacteria sustain plasmid replication?
Ori
EcoRINotI
HindIII
VP7
Uses of Expression Vectors in Genetic Research
Animal models of diseases (Various genetic techniques)Wide range: overexpressing genes to gene silencing
Genomic Studies
Gene therapy
There are many types of Expression Vectors that researcherscan choose from. Choice depends on the experiment.
Several properties of vectors are essential for function.
Features to have: 1. high level of expression2. tight control3. subtle modulation
Site-Directed Mutagenesis (Use of Vectors and PCR)
TCTATGGACCAGTACGATACCAGTA.....CGACCTACGTAGACTAGACGGATAGAG AGATACCTGGTCATGCTATGGTCAT.....GCTGGATGCATCTGATCTGCCTATCTC
GAATTCTCTATGGACCAGTACGATACCAGTA.....CGACCTACGTAGACTAGACGGATAGAGGGATCCCTTAAGAGATACCTGGTCATGCTATGGTCAT.....GCTGGATGCATCTGATCTGCCTATCTCCCTAGG
Addition of EcoRI(GAATTC) and BamHI (GGATCC) sitesand cloning into vector of choice
TCGATGGACCAGTACGATACC (mutant forward primer extended) 5’T C G ATGGACCAGTACGATACC----->extension AGATACCTGGTCATGCTATGGTCAT.....GCTGGATGCATCTGATCTGCCTATCTC
(First annealing is imperfect because 5’ end of primer is single stranded for 3 nt. After cycle 1, annealing is perfect through all 21 nt and the mutation is copied into product by extension from reverse primer).
TCGATGGACCAGTACGATACCAGTA.....CGACCTACGTAGACTAGACGGATAGAG AGCTACCTGGTCATGCTATGGTCAT.....GCTGGATGCATCTGATCTGCCTATCTC
Using PCR to Add Additional Restriction Sites
Enzymes do not have a high efficiency if they are perched at the end of the DNA molecule.
Enzyme Oligo Length Chain Length %CleavageBamHI CGGATCCG CGGGATCCCG
CGCGGATCCGCG
81012
109090
USES OF VECTORS TO GENERATE TRANSGENICS
MMTV-LTR Gene XVector Backbone
CGCGTTCTTGAAAAGACAGGTAAAATGCGAGTTCCAGAATGGGTAGAATTGTAAAGT
CTGCACGAT CCATATGATCCAGATTGGTATTATATTAGATGTGC
TGCTTTAGTTCGTCATATTTATATTCGAAG AGTAACAAAAA
TTTTTGGAGGACGCAAACGTAATGGTACTCATCCTAGCCATTTCTGTCGATCGGCAG
GTGGTGTTGCTCGCAAAGCTCTTCAGAGCTTGGAACAACTTAAACTCATTGAAAAATC
TCCAGTTGGTGGACGTA CGTAGAGATTTAGATCGCATTGC
CAAAAAACAACTTAAGTTACAAGAAACTCTTGTTCTC
WILD TYPE:
MUTANT 1:
MUTANT 2:
MUTANT 3:
MUTANT 4:
MUTANT 5:
MUTANT 6:
*
Cloning sitesOriAmpr
Vector Backbone
Excision/Purification
Implant embryos into oviductof surrogate mouse
Genotype progeny (potential founders)using Southern Blot or PCR
Identify founders (express transgene) to generate transgenic lines
Analyze phenotype, biochemical analyses of tissues,Cross transgenics with other transgenics (bigenics)
Integrate transgene into pronucleus of 1 cell embryo (RANDOM INTEGRATION)
Transgenic
Surrogate
Generating Knockout Mice using Expression Vectors in Embryonic Stem Cells
Generate Targeting Vector & transfect ES cells derived from blastocyst
Homologous recombination of genomic and targeting alleles
Random Recombination
Random recombination & negative selection
Injection of Genetically Engineered ES cells into Blastocysts
Neomycin + Ganciclovir
Knockout mouse
Epidermal growth factor receptor (Egfr)
Phenotype depends on genetic background: CF-1 background:
Degeneration of the inner cell mass Peri-implantation death
129/Sv background: Placental defects Death at mid-gestation
CD-1 background:
Abnormalities in skin, kidney, brain, liver, and gastrointestinal tract (Threadgill et al., 1995) Death by 3 weeks
129/Sv X C57BL/6 background: Death at birth
129/Sv X C57BL/6 X MF1 background: Death by postnatal day 20
Phenotypic abnormalities in newborn mice (Sibilia and Wagner, 1995)
Open eyes Rudimentary whiskers Immature lungs Defects in the epidermis
Database of Gene Knockouts
GENE KNOCKOUTS LISTED ALPHABETICALLY ACCORDING TO THE NAME OF THE GENE
The list can be scrolled through sequentially, or accessed by clicking any of the following letters:A B C D E F G H I J K L M N O P Q R S T U V W X Y Z
Example:
Embryonic Lethality How do we overcome
this limitation?
The End