1. Chapter 4: Basic Tools and Techniques of DNA Science (Part 2)
By Mitch Choi

2. 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)

4. 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

5. 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)

8. 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

9. 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

10. 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

11. 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”

12. 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

13. 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

14. 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

15. Escherichia Coli and Foreign DNA
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

16. 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

18. Bacterial Growth
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

19. 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 hours

20. 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

21. 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 𝑀𝑔 2+ , 𝑀𝑛 2+ , and 𝐵𝑎 2+ yielded similar or greater transformation efficiencis

22. 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

23. 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

25. 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

27. 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

28. 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

29. 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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