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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