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Chapter 4: Basic Tools and Techniques of DNA Science (Part
2)
By Mitch Choi
Section 1 - The Plasmid Vector
• Vector (Medical) – Organism that carries a pathogen from on host organism to another
• Vector (Molecular Biology) – A DNA molecule that is used as a vehicle to carry foreign DNA sequences into a host cell
• Plasmids are the simplest bacterial vectors
• Circular DNA molecules separate from main bacterial chromosome
• 1,000 to 200,000 base pairs
• Can be altered by scientists (for propagation or gene expression)
Propagation of Plasmids
• Bacteria grow rapidly
• Can produce large quantities of specific gene sequences
• Plasmid is placed into host cell
• Cell duplicates multiple times
• Amount of copies of gene sequences in plasmid grow exponentially
• Plasmid MUST contain specific DNA sequence – origin of replication (ori)
• Proteins required for DNA synthesis bind to ori
• Two groups of plasmids (depending on regulation of replication):
• Stringent Control – Replicate once per division with main chromosome
• Relaxed – Replicate DNA autonomously throughout cell cycle (hundreds of copies per cell, useful for amplifying large amounts of DNA)
Selectable Markers
• When using plasmids in experiments, not all cells contain the plasmid
• Must distinguish cells that do from cells that don’t
• Plasmids contain genes that encode selectable marker proteins
• Most common type is antibiotic resistance
• Host cells are grown in media containing antibiotics
• Only cells with the plasmid with resistance to the antibiotics will survive, while others will not
• Only plasmid containing cells are left
Inserting New Genes into Plasmids
• Gene cloning is essentially cutting and pasting DNA fragments into cells
• Key is to put a gene of interest into the plasmid vector
• Plasmid vectors are designed to have one or more “cloning sites”
• Cloning sites are a series of restriction recognition sequences for a variety of restriction endonucleases (enzymes) called a polylinker
• Restriction enzymes cut open the circular plasmid DNA and allows insert DNA to be spliced in
• The combination of two pieces of DNA is called a recombinant DNA molecule
• Many restriction enzymes create single-stranded overhangs (sticky ends) after cutting DNA
• Sticky end can for hydrogen bonds with complementary nucleotides from the sticky end of another fragment created from the same restriction enzyme
• The hydrogen bonds are weak and constantly form and break
• Holds the fragments long enough for DNA ligase to re-form the phosphodiester bonds
• Ligation maintains the double helical shape
• There are different ways to ligate DNA into vectors
• Restriction enzymes can cut out genes of interest and the fragments can be ligated into one plasmid vector
• Ideally, a gene is isolated by two DIFFERENT restriction enzymes on either side
• Each end has a different sticky end so the fragment doesn’t bond to itself
• Plasmid vector is then opened up with the same two restriction enzymes to create complimentary sticky ends for the fragment
• Called “directional cloning”
Review of Section 1
• A plasmid vector is a DNA molecule that is used as a vehicle to carry foreign DNA sequences into a host cell
• Plasmid vectors are used to create multiple copies of a gene or to have the gene expressed in a host cell
• Plasmid vectors have selectable markers to distinguish between which cells have them and which don’t
• New genes can be inserted into plasmids
Section 2 - The Host Cell: The Bacterium Escherichia Coli
• Propagation of genes must take place in a living cell
• Transformation of a cell is required for propagation
• Transformation – the cellular uptake and expression of DNA in a bacterium
• Requirements for efficient and useful transformation:
• 1. Suitable host organism to insert the gene in
• 2. A self-replicating vector to carry the gene into the host
• 3. A means of selecting for host cells that have taken up the gene
• E. Coli is the most widely used organism in molecular biology
• Provides simple and well-understood genetic environment to isolate foreign DNA.
• The E. Coli genome has been completely sequenced and more than 4,000 genes identified
• Genetic code is nearly universal, so E. Coli can accept foreign DNA from any organism
• All DNA is composed of adenosine, cytosine, guanosine, and thymidine and replicated the same way
• DNA is transcribed into mRNA, then translated into proteins
• Sometimes, E. Coli transcribes and translates foreign DNA the same way
Escherichia Coli and Foreign DNA
• Even under the best circumstances, the uptake of a specific foreign gene is rare
• Most easily accomplished through large populations of organisms that reproduce rapidly (like E. Coli)
• Recombinant plasmid is amplified when transformed bacteria replicate by binary fission
• Under favorable conditions, E. Coli replicates once every 22 minutes
• In 11 hours, 30 generations and 1 billion cells can be formed
• Each bacterium can carry up to several hundred copies of a cloned gene, resulting in the foreign DNA sequence being amplified by a factor of several hundred billion
• E. Coli inhabits the human colon, where it absorbs digested food materials
• For this reason, it grows best at 37° C in Leruia-Bertani (LB) broth, which contains carbohydrates, amino acids, nucleotide phosphates, salts, and vitamins
• Bacteria grow in several distinct phases:
• Lag phase – cells adjust to nutrient environment and prepare for rapid proliferation
• Logarithmic (log) phase – culture grows exponentially
• Stationary phase – Cell number becomes constant as new cells are produced, while older cells die
• Death phase – nutrients deplete, wastes accumulate, and bacteria die
Bacterial Growth
• Masses of bacterial cells are grown in a suspension culture
• To isolate individual colonies, cells are spread onto the surfaces of LB agar plates
• Eventually, the cells divide to form visible daughter colonies after 12-24 hours
Section 2 Review
• E. Coli is commonly used for the propagation of genes because it is a well understood bacteria
• E. Coli can accept foreign DNA from any organism
• Bacteria have four phases
• Bacteria culture in LB media
Section 3 - Transformation• Transformation is a tool for manipulating the genetic makeup of living things
• In 1970, Morton Mandel and Akiko Higa found that E. Coli becomes markedly competent for transformation by foreign DNA when cells are suspended in cold calcium chloride solution and subjected to a brief heat shock at 42° C
• Cells arrested in early to mid-log growth are even more competent
• The calcium chloride procedure yields efficiencies of to transformants per microgram of plasmid DNA
• Treatment with other cations such as and yielded similar or greater transformation efficiencis
• Under conditions that yield high-efficiency transformation, approximately 1/10 of all viable cells are competent
• At an efficiency of transformants per microgram of plasmid, only 1/100,000 of the cells become competent
• Size and conformation of the DNA molecule affect transformation efficiency
• Small plasmids are more readily taken up than larger ones, however no preferred size cutoff is evident
• DNA molecules are too large to diffuse or be readily transported through the cell membrane
• Some bacteria possess membrane proteins that recognize DNA and facilitate the absorption of short DNA sequences
• One hypothesis is that DNA molecules pass through any of several hundred channels at adhesion zones
• Adhesion zones are only present in growing cells, supporting the observation the cells in the log phase are more competent
• However, the phosphates of the DNA and phospholipids are negatively charged, causing repulsion
• Treatment at 0° C causes fluid in the cell membrane to freeze
• Cations are formed to shield the anions
• Heat shock creates a thermal imbalance on either side of the E. Coli membrane that pumps the DNA through the adhesion zone
Section 4 - Electroporation
• Large circular DNA molecules are too large to be taken up by cells naturally
• To insert large DNA molecules, electroporation is used
• Cells are grown to late log phase and washed thoroughly in ion-free water
• Cells and vector are placed into a small chamber with metal sides (electrodes)
• Pulse of electricity is applied and alters the electrical properties of the cells, allowing DNA to enter
• No theoretical size limit
• Can be used on living animals by injecting the plasmid next to target cells and generating a current
Section 5 – Isolation and Analysis of Recombinant Plasmids
• Growth of colonies on antibiotic media provides phenotypic evidence of transformation
• To confirm at the genotypic level, plasmid DNA is isolated from transformants
• Restriction analysiz of the purified plasmid DNA with the original DNA used to make the recombinant prove the genetic identity
DNA Miniprep Procedure
• Miniprep – rapid method for making a small preparation of purified plasmid DNA from culture volumes as low as 1 mL
• Transformed cells from an antibiotic-resistant colony are grown to stationary phase
• Cells collected by centrifugation and kept in a solution of glucose and EDTA for membrane stability
• Cells are treated with SDS (ionic detergent) and sodium hydroxide to dissolve phospholipids and proteins, releasing cell contents
• Sodium hydroxide denatures plasmid and chromosomal DNAs into single strands
• Treatment with potassium acetate and acetic acid forms insoluble precipitate of SDS/lipid/protein and neutralizes sodium hydroxide
• Chromosomal DNA partially renatures and gets trapped in precipitate, plasmids completely renature
• Cetrifugate and discard precipitate
• Add ethanol or isopropanol to supernatant to precipitate plasmids out of solution
• Pelleted by centrifugation
• Washed with ethanol, dried, resuspended in buffer
• Treatment with RNase, destroying RNA, leaving clean plasmid DNA
• Original plasmid and miniprep plasmids are run on agarose gel and compared
• Larger scale preparations called maxipreps
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