Transformation and selection of recombinant bacteria
Content
Introduction ......................................................................................................................................................... 2
Materials for the transformation ....................................................................................................................... 5
Thawing the cells ............................................................................................................................................... 6
Adhesion of DNA to the cells ........................................................................................................................... 6
Heat shock .......................................................................................................................................................... 7
Recovering of cells ............................................................................................................................................ 9
Antibiotic selection and blue/white screening .............................................................................................. 10
Materials for selection of recombinant bacteria .......................................................................................... 12
Platting of cells (1) ........................................................................................................................................... 13
Concentration of cells...................................................................................................................................... 14
Platting of cells (2) ........................................................................................................................................... 14
Incubation of cells ............................................................................................................................................ 14
Result of a blue/white screening ................................................................................................................... 15
Protocol ............................................................................................................................................................. 16
1
Transformation and selection of recombinant bacteria
Introduction
Transformation (fig.1) is the directed modification of the genome of a cell by the external
application of purified or recombinant DNA from another cell of a different genotype. In general it is
possible to integrate DNA of any species into the genome of another one. The term transformation
though is mostly used to specify the uptake and incorporation of exogenous DNA into bacteria.
Introducing DNA fragments into eucariotic cells is called transfection (fig. 2). Introducing DNA of
another species via a virus or bacteriophage into a cell is also called transduction (fig. 3). As the
term transformation, the term transduction is mostly used to describe the insertion of DNA into
bacterial cells.
Fig. 1: Transformation
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Transformation and selection of recombinant bacteria
Fig. 2: Transfection
Fig. 3: Transduction
3
Transformation and selection of recombinant bacteria
Before a transformation of bacteria with purified DNA of any species, the DNA fragment of interest is
introduced into a plasmid vector via a process called ligation. The ligated plasmid is then called
recombinant plasmid (fig. 4).
Fig. 4: Ligation: A plasmid vector, which has been cleaved by a restriction enzyme and
a DNA fragment are covalently linked. The reaction is catalysed by the enzyme
ligase.
Usually nucleic acids, such as plasmids do not enter bacteria under their own power, but require
assistance traversing outer and inner cell membranes. Therefore bacteria have to be treated before
transformation. A variety of methods exist to achieve this goal. One possibility is to use chemically
induced competent cells, also called salt competent cells. These cells have been treated with
Calcium Chloride (CaCl2).
4
Transformation and selection of recombinant bacteria
Materials for the transformation
For the transformation of bacteria you need …..
2
2
3
4
6
1
5→
7→
Fig. 5: Materials for transformation of bacteria
1 a bucket with ice
2 two water baths or incubators for the transformation process and the pre-cultivation of the
transformed bacteria
3 a clock
4 an autoclavable flask for fluid bacteria waste
5 approx. 100 – 200 µl of a competent bacteria culture (kept on ice)
6 liquid broth
7 recombinant plasmid DNA
And, not shown
- sterile 1,5 ml reaction tubes
- a set of micropipettes and sterile tips for amounts in between 200 and 1000 µl. Sterile tips should
be used to avoid contamination of the isolated DNA and the bacteria.
Competent cells are fragile and should be handled gently. Therefore always keep them on ice. Do
not vortex the cells. When pipetting competent cells use tips with large diameters.
5
Transformation and selection of recombinant bacteria
Thawing the cells
1. Salt competent cells are usually stored in a freezer at minus 80 °C or in liquid nitrogen. Is this the
case thaw the competent cells for 15 minutes on ice.
→
Fig. 6: Thawing competent cells (→) on ice
Adhesion of DNA to the cells
2. Add a volume of 10 µl (approx. 1 to 10 ng of DNA) to 100 - 200 µl of competent cells.
Mix gently.
3. Store the tube on ice for another 30 minutes.
This allows the adhesion of the DNA to the cells.
Fig. 7: Adding of recombinant DNA to the competent cells
6
Transformation and selection of recombinant bacteria
Heat shock
4. Transfer the tube into a preheated incubator of 42°C for one to two minutes.
Do not shake the tubes.
This treatment is called heat shock. The high temperature increases the movement of the
membrane-phospholipids: The DNA may enter the cell (see fig. 10).
5. After two minutes rapidly transfer the tubes to the ice bath in order to avoid damage to the cells.
6. Allow the cells to cool for 5 minutes on ice.
Fig. 8: The bacteria are transferred into a preheated
incubator of 42°C for the heat shock.
Fig. 9: After the heat shock the bacteria are transferred into an
ice bath.
7
Transformation and selection of recombinant bacteria
B
C
A
Fig. 10: Heat shock: Either no DNA at all (A) non recombinant plasmid DNA (B) or recombinant plasmid
DNA (C) may enter the cell. In a process called selection recombinant bacteria will be selected
(see chapter “Antibiotic selection and blue/white screening”).
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Transformation and selection of recombinant bacteria
Recovering of cells
7.
8.
After 5 minutes of cooling add 1000 µl of liquid media without any antibiotics to the tube.
Mix liquid and bacteria gently.
To help the bacteria recover from the heat shock, the cells are incubated with non-selective growth
media. As the cells recover, plasmid genes are expressed, including those that enable the
production of daughter plasmids which will segregate with dividing bacterial cells.
9.
Transfer the culture to a 37°C preheated incubator. Do not shake the tubes for the first 15
minutes.
10. Incubate for further 45 minutes while gently shaking at approximately 600 revolutions per
minute.
Fig. 11: Add liquid media to the cells after the heat shock …
.
Fig. 12: … and place them in an incubator,
Start shaking after 15 minutes.
After one hour of growing in non selective liquid media the cells are ready for selection.
9
Transformation and selection of recombinant bacteria
Antibiotic selection and blue/white screening
Usually the transformation rate of chemically induced competent cells is very low. The
transformation rate may not be higher than one in 10,000. It is necessary to select for bacterial cells
which contain plasmids (see fig. 9).
This is commonly performed with antibiotic selection. Many E. coli strains are sensitive to antibiotics.
Plasmids used for the cloning and manipulation of DNA have been engineered to harbor the genes
for antibiotic resistance. Thus, if the bacteria after transformation are plated onto nutrient agar plates
containing antibiotics, only bacteria which posses the plasmid DNA will have the ability to survive
and form colonies. In this way, bacterial cells containing plasmid DNA are selected.
In order to isolate cells containing recombinant plasmids from those containing non-recombinant
plasmids, the E. coli lacZ´ gene has been incorporated into several plasmid vectors (see fig. 13).
These plasmids carry a fragment of the gene for β-galactosidase - also known as lacZ`. This
enzyme is part of a set of enzymes which catalyze the breakdown of lactose as a food source.
These enzymes will only be expressed, if lactose but no glucose is present in the growth media.
Plasmid vectors designed for blue/white screening have a multicloning site carefully placed within
the coding region of lacZ´. Successful ligation of a foreign DNA into this multicloning site interrupts
the gene and abolishes production of functional β-galactosidase.
B
A
Fig. 13: Plasmid vector for blue/white screening
A: non recombinant plasmid: the foreign DNA fragment has not been
transferred into the plasmid, lacZ` is not interrupted.
B: recombinant plasmid: the foreign DNA fragment has been transferred into
the plasmid, lacZ` is interrupted.
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Transformation and selection of recombinant bacteria
For the selection two reagents, besides an antibiotic are added to nutrient agar plates on which the
transformed cells are plated:
- IPTG (isopropylthiogalactoside)
- and X-gal (5-bromo-4-chloro-indolyl-β-D-galactopyranoside).
IPTG is an inducer for the gene β-galactosidase, meaning the enzyme will be expressed if the
reagent IPTG is present. X-gal is a colourless lactose analogue and chromogen. It turns blue
when hydrolysed by β-galactosidase and exposed to oxygen. The blue-colored product is
precipitated within the cells.
Not transformed cells will not grow at all due to the lack of antibiotic resistence. Transformed but
non-recombinant cells will turn blue due to the presence of non recombinant plasmids.
Recombinant cells will grow, but keep their natural color, due to the interrupted lacZ´ gene, which
can not be expressed (see fig. 14).
Of course the blue/white screening can only be performed with E.coli strains having a deletion of the
lacZ within their chromosome.
A
B
C
Fig. 14: The blue white screening
A: non transformed bacterium (non transformant)
B: transformed but not recombinant bacterium
C: recombinant bacterium
11
Transformation and selection of recombinant bacteria
Materials for selection of recombinant bacteria
For the selection you need …
8
3
7
2
5→
8
6
1
4
Fig. 15: Materials for selection of recombinant bacteria
1
2
3
4
5
6
7
8
a clean bench
a gas burner
a micropipette
sterile tips for amounts in between 50 - 100 µl
a drigalski spatula
70 % ethanol
transformed cells in liquid media (on ice)
and in case of the blue/white screening: nutrient agar plates containing an antibiotic and the
reagents IPTG and X-gal.
And not shown:
- an autoclavable flask for fluid bacteria waste
- a table top centrifuge in order to pellet the bacteria culture
- an incubator for agar plates.
12
Transformation and selection of recombinant bacteria
Platting of cells (1)
In case of the blue/white screening the cells are plated on nutrient agar plates containing an
antibiotic and the reagents IPTG and X-gal.
1. Sterilize the drigalski spatula by bathing it in ethanol of a percentage of 70 % and flaming it.
When working near an open flame never wear latex or nitrile gloves. Severe burnings may
occure.
2. Allow the spatula to cool.
3. Mix the precultivated bacteria gently and pipette 100 µl of the suspension on a nutrient agarplate
4. Spread the bacteria carefully over the complete agar surface using the sterilized and cooled
drigalski spatula. The liquid should completely diminish into the agar plate.
5. After plating sterilize the spatula.
6. Do not forget to label the plates on the bottom of the plate.
Fig. 16: Sterilisation of the drigalski spatula
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Transformation and selection of recombinant bacteria
Concentration of cells
To increase the concentration of the cells
7. Centrifuge the remaining suspension 1 minute with 13000 rpm.
8. In order to stabilize the centrifuge, do not forget to add a tube of the same weight opposite of
the tube containing the bacteria,
9. Remove approximately 900 µl of the liquid media and resuspend the bacteria pellet in the
remaining liquid.
Fig. 17: The removed liquid media is transfered into the
autoclavable flask for liquid bacterial waste.
Platting of cells (2)
10. The remaining bacteria are also plated on nutrient agar plates containing an antibiotic and the
reagents IPTG and X-gal (see “plating of cells (1)”).
Incubation of cells
11. Store the plates for a couple of minutes at room temperature until the liquid has been
completely absorbed.
12. Invert the plates and incubate them at 37 °C. Inverting the plates will avoid dripping of
condensate on the growing bacteria.
13. Transformed cells should appear within 12 – 18 hours.
14
Transformation and selection of recombinant bacteria
Result of a blue/white screening
The plate below shows the result of the blue/white screening (see fig. 18):
Only bacteria resistant against the antibiotic have grown.
The blue cells carry non recombinant plasmids: The gene lacZ´ has not been interrupted by the
foreign gene. Its expression was induced by IPTG. The chromogen X-gal was hydrolysed and turned
blue. The grayish colonies are the recombinants. These E. coli cells carry recombinant plasmids.
Fig. 18: Result of a blue/white screening
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Transformation and selection of recombinant bacteria
Protocol
Transformation of bacterial cells
Materials
- a bucket with ice
- two water baths or incubators for the transformation process and the pre-cultivation of the
transformed bacteria
- a clock
- an autoclavable flask for fluid bacteria waste
- approx. 100 – 200 µl of a competent bacteria culture (kept on ice)
- liquid broth
- recombinant plasmid DNA
- sterile 1,5 ml reaction tubes
- a set of micropipettes and sterile tips for amounts in between 200 and 1000 µl
Competent cells are fragile and should be handled gently. Therefore always keep them on ice. Do
not vortex the cells. When pipetting competent cells use tips with large diameters.
Thawing the cells
1. If the competent cells have been stored in a freezer at minus 80 °C or in liquid nitrogen, thaw the
competent cells 15 minutes on ice.
Adhesion of DNA to the cells
2. Add a volume of 10 µl (approx. 1 to 10 ng of DNA) to 100 - 200 µl of competent cells.
Mix gently.
3. Store the tube on ice for another 30 minutes.
Heat shock
4. Transfer the tube into a preheated incubator at 42°C for one to two minutes.
Do not shake the tubes.
5. After two minutes rapidly transfer the tubes to the ice bath.
6. Allow the cells to cool for 5 minutes on ice.
Recovering of cells
7. Add 1000 µl of liquid media without any antibiotics to the tube.
8. Mix liquid and bacteria gently.
9. Transfer the culture to a 37°C preheated incubator. Do not shake the tubes for the first 15
minutes.
10. Incubate for further 45 minutes by gently shaking at approximately 600 revolutions per minute.
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Transformation and selection of recombinant bacteria
Selection of recombinant cells
Materials
a clean bench
a gas burner
a micropipette and sterile tips for amounts in between 50 - 100 µl
a drigalski spatula
70 % ethanol
transformed cells in liquid media (on ice)
nutrient agar plates: in case of the blue/white screening, containing an antibiotic and the
reagents IPTG and X-gal
an autoclavable flask for fluid bacteria waste
a table top centrifuge in order to pellet the bacteria culture
an incubator for agar plates
Plating of cells (1)
1. Sterilize the drigalski spatula by bathing it in ethanol of a percentage of 70 % and flaming it.
2. Allow the spatula to cool.
3. Mix the precultivated bacteria gently and pipette 100 µl of the suspension on a nutrient agarplate
4. Spread the bacteria carefully over the complete agar surface using the sterilized and cooled
drigalski spatula. The liquid should be completely diminish into the agar plate.
5. After plating sterilize the spatula.
6. The plates should be labelled on the bottom.
Concentration of cells
7. Centrifugate the remaining suspension 1 minute with 13000 rpm.
8. In order to stabilize the centrifuge, do not forget to add a tube of the same weight opposite of the
tube containing the bacteria,
9. Remove approximately 900 µl of the liquid media and resuspend the bacteria pellet in the
remaining liquid.
Plating of cells (2)
10. The remaining bacteria are also plated on nutrient agar plates containing an antibiotic and the
reagents IPTG and X-gal (see “plating of cells (1)”).
Incubation of cells
11. Store the plates for a couple of minutes at room temperature until the liquid has been completely
absorbed.
12. Invert the plates and incubate them at 37 °C. Inverting the plates will avoid dripping of
condensate on the growing bacteria.
13. Transformed cells should appear within 12 – 18 hours.
17