Lab2-Plasmid Isolation (1)

8/19/2019 Lab2-Plasmid Isolation (1)

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Microbial Genetics (GEN 203)

Lab Two

Plasmid Isolation

Course Coordinator: Dr. Ashraf Bakkar

L.As: Ingy El-Hefny, Heba Hisham , T.A: Aya Farghally


2/15

What are plasmids?

• A plasmid is distinct or independent from a cell’s

chromosomal DNA.It’s main characteristics are:

small and circular

double-stranded DNA molecule

Self replicating

• Plasmids naturally exist in bacterial cells only, right?

• WRONG! They can occur in some eukaryotes too such as

Entamoeba histolytica, yeast etc.


3/15

1) The bacterial chromosome: DNA of most bacteria is contained in

a single circlar molecle, called the bacterial chromosome.2) The nucleoid: is an irreglar sha!e that contains se"eral !roteins

and #NA molecles

3) Plasmids: small circlar DNA molecles.


4/15

Types of Plasmids

• Plasmids are not usually required by their host cell for its survival.

Instead, they carry genes that confer aselective advantage on their host.

• Plasmids are classified into five main types according to that

ADVANTAGE:

1.Fertility F-plasmids:which containstra genes. They provide the

fertility system required for conjugation. Sometimes they are called

episomes.

2.Resistance (R)plasmids:R plasmids carry genes encoding resistance

to naturally made antibiotics by other microorganisms or to toxic

substanaces such as heavy metals


5/15

3. Col plasmids:Col plasmids allows their host to produce

antibacterial polypeptides called bacteriocins that are often

lethal to closely related or other bacteria. The col proteins ofE.

coli are encoded by plasmids such as ColE1.

4. Degradative plasmids: allow a host cell to metabolize

normally undegradable or difficult compounds such as

various pesticides.

5. Virulence plasmids:which turn the bacterium into a pathogen


6/15

How can we isolate plasmids?

• First:we isolate plasmids from the bacterial cells bythe

alkaline lysis methodwhich depends on a unique property

of plasmid DNA; it’s ability to rapidly anneal following

denaturation. This is what allows the plasmid DNA to be

separated from the bacterial chromosome.

• Second: we purify it from the rest of the cellular components

bythe silica based membrane technology


7/15

Alkaline Lysis

1. Harvest bacterial Cells: Bacterial cells are pelleted by centrifugation to

remove them from the growth medium.

2. Re-suspension: The pellet is then re-suspended in a solution containing

often Tris, EDTA, and RNase A.

Function of:

A)EDTA:chelates divalent cations such as (Mg2+, Ca2+) in the solution

preventing DNases from damaging the plasmid and also helps by

destabilizing the cell wall.

B) RNase A:to degrade cellular RNA when the cells are lysed.


8/15

3. Lysis solution:contains sodium hydroxide (NaOH) and the detergent Sodium

Dodecyl (lauryl) Sulfate (SDS).

Function of:

A)SDS:is there to solubilize the cell membrane and denatures proteins.

B) NaOH:helps to break down the cell wall, but more importantly it denatures

DNA by disrupting the hydrogen bonding between the DNA bases, converting

the double-stranded DNA (dsDNA) in the cell, including the genomic DNA

(gDNA) and your plasmid, to single stranded DNA (ssDNA). This is why it’s

calledalkaline lysis.


9/15

4. Neutralization solution:contains potassium acetate

Potassium acetate has two functions:

1)Re-naturation of plasmids:it decreases the alkalinity of the mixture, so

the hydrogen bonding between the bases of the single stranded DNA can

be re-established. It is only easy for the small circular plasmid DNA to

re-nature.

2)Precipitates unwanted cell debris and gDNA:large ssDNA are insoluble

in high salts, while the double-stranded plasmid can dissolve easily in

solution. As a result the single stranded genomic DNA, the SDS and the

denatured cellular proteins will form a white precipitate.


10/15

Now your plasmid DNA has been separated from themajority of the cell debris but is in a solution

containing lots of salt, EDTA, RNase and residual

cellular proteins and debris, so it’s not much use

for downstream applications.

The next step is to clean up the solution and

concentrate the plasmid DNA.


11/15

Silica Membrane Based Technology

• Principle:

• Chaotrophic salts are added to the sample to denature the DNA by disrupting it’s

hydrogen bonding.

• Under these conditions, the DNA will selectively bind to the silica resin because

hydrogen bonds will form between the membrane and the phosphate backbone.

• After washing the DNA is eluted

from the column with a low salt

solution which allows the re-

naturing of the DNA, causing it to

lose affinity for the silica.


12/15

Smmary


13/15

!ow yor plasmid is ready for downstream

applications


14/15

NEXT WEEK

• Possible troubleshoots

• Detect your plasmid!

• Different Plasmids Structure and plasmid

Replication• Difference between Plasmids and Vectors


15/15

THA!" #$%

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Lab2-Plasmid Isolation (1)