EXPERIMENT 5 &6

“PLASMID TRANSFORMATION”
“ISOLATION OF PLASMID DNA FROM BACTERIAL
CELLS”

INTRODUCTION AND AIM

Transformation is the genetic alteration of a cell resulting from the introduction, uptake and
expression of foreign genetic material (DNA or RNA). It is a common technique in molecular
biology.

In Bacterial Transformation, it refers to a genetic change brought about by taking up and

expressing DNA. As we know genetic materials are not very small, sometimes different
methods can be applied to bacteria to transform genetic material to its cytoplasm. Accepting a
new DNA, RNA or plasmids is about being competent. Competence refers to the state of
being able to take up exogenous DNA from the environment. Two different forms of
competence should be distinguished: natural and artificial.

Natural competence: Some bacteria (around 1% of all species) are naturally capable of
taking up DNA under laboratory conditions; many more may be able to take it up in their
natural environments. Such species carry sets of genes specifying machinery for bringing
DNA across the cell's membrane or membranes.

Artificial competence: Artificial competence is not encoded in the cell's genes. Instead it is
induced by laboratory procedures in which cells are passively made permeable to DNA, using
conditions that do not normally occur in nature. Mostly 2 techniques are used to make
competent cells.

• Chilling cells in the presence of divalent cations

• Electroporation

Chilling cells in the presence of divalent cations such as CaCl2 prepares the cell walls to
become permeable to plasmid DNA. Cells are incubated with the DNA and then briefly heat
shocked (42°C for 30-120 seconds), which causes the DNA to enter the cell. This method
works well for circular plasmid DNAs but not for linear molecules such as fragments of
chromosomal DNA probably because exonuclease enzymes in the cell rapidly degrade linear
DNA. Cells that are naturally competent are usually transformed more efficiently with linear
DNA than with plasmids.

Electroporation is another way to make holes in bacterial (and other) cells, by briefly
shocking them with an electric field of 10-20kV/cm. Plasmid DNA can enter the cell through
these holes. This method is amenable to use with large plasmid DNA Natural membrane-
repair mechanisms will rapidly close these holes after the shock.

Plasmid is a DNA molecule separate from the chromosomal DNA and capable of
autonomous replication. It is typically circular and double-stranded. It usually occurs in
bacteria, sometimes in eukaryotic organisms. There may be one copy, for large plasmids, to
hundreds of copies of the same plasmid in a single cell, or even thousands of copies, for
certain artificial plasmids selected for high copy number.

Another marker, used for identifying E. coli cells that have acquired recombinant plasmids, is
the lacZ gene, which codes for β-galactosidase. Because β-galactosidase is a homo-tetramer,
with each monomer made up of one lacZ-α and one lacZ-ω protein, if only one of the two
requisite proteins is expressed in the resulting cell, no functional enzyme will be formed.
Thus, if a strain of E. coli without lacZ-α in its genome is transformed using as plasmid
containing the missing gene fragment, transformed cells will produce β-galactosidase, while
untransformed cells will not, as they are only able to produce the omega half of the monomer.
In this type of transformation, the polylinker region of the plasmid lies in the lacZ-α gene
fragment, meaning that successfully produced recombinant plasmids will have the desired
gene inserted somewhere within lacZ-α. When this disrupted gene fragment is expressed by
E. coli, no usable lacZ-α protein is produced, and therefore no usable β-galactosidase is
formed. When grown on media containing the colorless, modified galactose sugar X-gal,
colonies that are able to metabolize the substrate (and that have therefore been transformed,
but not by recombinant plasmids) will appear blue in color; colonies that are not able to
metabolize the substrate (and that have therefore been transformed by recombinant plasmids)
will appear white.

In this experiment our aim is adding a plasmid which contains pGEM, was produced by
combining antibiotic resistance gene for ampicilline (ampI), to our bacteria E.coli JM109 to
make them resistant to ampicilline. And also we transform a part of gene to bacteria, because
we want them to produce β-galactosidase. At the second part of the experiment we
differentiate the bacterial chromosomes and plasmid we added (pGEM) from the bacteria we
produce (resistant to ampicilline). The method we will use is alkaline lysis method. Alkaline
lysis is a method used in molecular biology to break cells open to isolate plasmid DNA or
other cell components such as proteins. Bacteria containing the plasmid of interest is first
grown, then lysed with a strong alkaline buffer consisting of a detergent sodium dodecyl
sulfate (SDS) and a strong base sodium hydroxide. The detergent breaks the membrane's
phospholipid bilayer and the alkali denatures proteins involved in maintaining the structure of
the cell membrane. Through a series of steps involving agitation, precipitation, centrifugation,
and the removal of supernatant, cellular debris is removed and the plasmid is isolated and
purified
MATERIALS AND METHODS

Transformation Isolation of Plasmid

• Agar plate • Micro pipette
• Glass rod • Tip
• Bunsen burner • Zephiran
• Ethanol (95 %) • Eppendorf tubes
• Micro pipette • Ice bucket
• Tip • PKS2
• Zephiran • D1
• Incubator • Glover
• Eppendorf tubes • Solution A, B, C
• Ice bucket • Vortex mixer
• Water bath • Absolute Ethanol
• PKS2 • 70 % Ethanol
• D1 • Rack
• SOC Medium • Centrifugal Machine
• E. Coli JM109 • Phenol Chloroform
• Glove • Paper Towel
• LB Media • Q-tip
• TE
Transformation methods

• First label 3 eppendorf tubes as “-”, “PKS2” and “D1 given to you by assistants. They
contain E. coli cells, plasmids and constructs.
• Transfer 100 µl competent cell to each tube you labeled.
• Add 10 µl plasmid DNA to tube 2 (labeled as PKS2) and 10 µl construct to 3rd
tube(labeled as D1)
• Keep all three tubes on ice for 30 minutes.
• Place tubes into a water bath and keep at 42°C for 1-2 minutes (do not shake)
• Rapidly transfer tubes to the ice bucket and chill tubes for 1-2 minutes.
• Add 900 µl SOC medium to each tube.
• Place cells in water bath at 37°C for 1 hour to allow bacteria to recover themselves.
• Add 150 µl from each tube on a separate agar plate which contain ampicilline, X-Gal
and IPTG
• Incubate tubes at 37°C overnight

Isolation methods

• We had 1.5ml tube of D1 and PKS2 culture which is grown at 37 centigrade degree.

• Centrifuge those 12000g. It is important that putting them facing one another in
order to balance tubes

• After centrifugation heavy materials sink bottom of the tubes. We poured out some
of the supernatant, used micro pipette to pour out rest of it.

• Add 100µl Solution A both tubes. Solution A has glucose, EDTA, and Tris in it.
Glucose maintains the osmotic pressure, EDTA weakens the cell membrane and
chelates Mg+2 that are needed by DNAase protecting the DNA

• Vortex the mixture the pellet with solution A.

• Later than vortexing this time we added 200µl Solution B. The aim of adding
solution B is rendering DNA double to single stranded. It has SDS and NaOH in it.
SDS dissolves the cell membrane lipids and degrades the cellular proteins. And the
NaOH splits the double stranded DNA into single stranded.

• This step is neutralization step

In this step we added 150µl solution C and vortexed gently.

 Solution C contains potassium acetate and ethanoic acid. Ethanoic acid neutralize
the solution enabling to DNA denature. The potassium acetate precipitates SDS,
proteins, lipids and large DNA molecules.
The E-coli chrosomal DNA was also trapped in the precipitate at this step. The
plasmid DNA remained in solution.

• After adding solution C, centrifuge them in 4 centigrade degree for 5 minutes.

Supernatant contained plasmid DNA, pellet contained cell debris.

• Take 300µl of supernatant and added 200µl phenol chloroform. Then again sent
them to be centrifuged for 2 minutes.

• That time, transferred the supernatant to new tubes. In the new tubes we added 2
volumes of absolute ethanol to solve the plasmid DNA. Because DNA is insoluble in
ethanol, ethanol precipitated DNA as well as the salts that form ionic bonds in it.

• Vortex final mixture and wait about two minutes at room temperature.

• Pellet the DNA by centrifugation for 5 minutes at 4 centigrade degree.

• Add 100µl 70 % ethanol.

• Decant the liquid from the tube. Remove any remaining liquid using micropipette.

• Invert the tube on a paper towel and drain for several minutes.

• Add 20µl TE to each tube for conserving the plasmids more time.
 RESULT

Isolation result

After applying all procedure finally we have pure genetic materials from our cells.

Transformation Result

• We have 3 different agar plates as you see below.

• First one does not contain any plasmid

• Second contains plasmid (+) (PKS2)

• And third one contains construct (+) (D1)

Plasmid (-) Plate Plasmid (+) Plate Construct (+) plate

No colony 132 blue colonies 89 white colonies

DISCUSSION

Transformation

• In first plate we do not expect to grow any bacteria culture because agar medium has
ampicilline and our bacteria in first plate does not have any resistance to ampicilline.
So the result overlaps our expectations.

• In second plate the colonies are blue. Bacteria in second plate can synthesis β-
galactosidase enzyme. This β-galactosidase converts the X-Gal to 5-bromo-4-chloro-
3-hydroxyindole and galactose by oxidizing. Normally our bacteria do not have the
gene that synthesis β-galactosidase.

They have only C terminal of LacZ gene. Because our plasmid consists of the other
part of the gene, N terminal, bacteria can produce β-galactosidase. This means our
bacteria have a functional LacZ gene. Galactose is one of the products of the reaction
above, and as a result of this producing galactose this colony became blue.

This means that we can transform the plasmid to the bacteria correctly. The results
conformed to our expectations one to one.

• In third plate we have D1 construct. Because this construct does not have N terminal
of the Lac Z gene, bacteria in the third plate cannot have an ability to produce β-
galactosidase. So their colonies color is white.

Surprisingly we have some of blue colonies in the plate. I don’t have any idea about
,what happened but I guess is because of the tips. When we were using micro pipette
we may forget changing the tips. As a result a little amount of plasmid can mix our
construct.

The result of this part does not overlap our expectations exactly because of the blue
colonies in the third plate.
Isolation

At the end of the experiment we have two tubes of pure genetic material. One of them is
plasmid and the other is construct. These materials place the bottom of the eppendorf tubes.

Actually, although we can see plasmids easily, we cannot observe any exact construct in
eppendorf tube. The tubes bottom are a little decayed and this causes us to make mistake
about the existence of construct.

There is nothing something to discuss anymore because we will see that our experiment is
failed or not next week after the agarose gel experiment.
REFERENCES

Lab Manual
http://en.wikipedia.org/wiki/Transformation_%28genetics%29
http://en.wikipedia.org/wiki/Plasmid
http://en.wikipedia.org/wiki/Alkaline_lysis