AIM:
To prepare competent cells which are capable of taking up foreign plasmid.
PRINCIPLE:
Transformation process allows a bacterium to take up genes from its surrounding environment; that is transformation involves the direct uptakes of fragments of DNA by a recipient cell and the acquisition of new genetic characteristics.
There are two major parameters involved in efficiently transforming a bacterial organism. The first is the method used to induce competence for transformation. The second major parameter is the genetic constitution of the host strain of the organism being transformed. Competent cells are capable of taking up DNA from their environment and expressing DNA as functional proteins. If a bacterium is said to be competent, it has to maintain a physiological state in which it can take up the donor DNA. Competence results from alterations in the cell wall that makes it permeable to large DNA molecules.
Transformation & Storage Solution (TSS) enables researchers to
prepare competent E. coli in a single step and to transform the cells without heat-shock. TSS has been reported to be faster and easier than other methods of producing competent cells, such as the traditional CaCl2 method
or other high-competency protocols.
The CaCl2 method
initially produces highly competent E. coli cells, but cell
competency decreases rapidly after storage at -70°C for several weeks. Other
protocols produce highly competent cells that have a long storage life, but the
procedures are time-consuming, requiring several transformation buffers or
heat-shock steps. In contrast, TSS is a simple, one-step procedure.
Advantages of using TSS
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In the process of transformation, the competent cells are incubated with DNA in ice. Then it is placed in a waterbath at 42ºC and further plunging them in ice. This process will take up the DNA into the bacterial cell. Then it is plated in an agar plate containing appropriate antibiotic.
The presence of an antibiotic marker on the plasmid allows for rapid screening of successful transformants. Blue –white selection (Alpha complementation) can be used to determine which plasmids carry an inserted fragment of DNA and which do not. These plasmids contain an additional gene (lac Z) that encodes for a portion of the enzyme β – galactosidase. When it transformed into an appropriate host, one containing the gene for the remaining portion of β –galactosidase, the intact enzyme can be produced and these bacteria form blue colonies in the presence of the chromogenic substrate X – gal (5-bromo-4-chloro-3-indoyl-b-D-galactoside).
These plasmids contains a number of cloning sites within the lac Z gene, and any insertion of foreign DNA into this region results in the loss of the ability to form active β –galactosidase. Therefore colonies that carry the plasmid with an insert (Transformants) will remain white and the colonies without the foreign DNA (Non-Transformants) will remain Blue.
We can also calculate the efficiency of transformation by using the
concentration of DNA and number of transformed colonies.
MATERIAL
REQUIRED:
TSS solution (transformation and
storage solution)
LB – 2.12gms
PEG – 10gms
100% DMSO – 5ml
1M MgCl2 (pH 6.5) – 5ml
Weigh these components, dissolve it completely in 80ml of milliQ water and make up the volume to 100ml with milliQ water. Autoclave and store at 40c (storage period: 1 week)
LB medium:
Weigh 2.5gms in 80 ml Milliq water in 500ml conical flask (head space is necessary for aeration). Dissolve it completely and make up the volume to 100ml with milliq water. Autoclave and store at room temperature.
Autoclaved dry 1.5ml eppendrof vials, sterile centrifuge bottles (250ml), dry ice with 100% ethanol.
PROCEDURE:
1. A
single colony (DH50 / sbl3) was inoculated into 5ml LB media and grown
overnight at 370C with shaking at 200rpm.
2. 1ml
overnight culture was inoculated into 500ml conical flask containing 100ml of
sterile LB broth.
3. Culture
was incubated at 370c/200rpm till it reaches an OD of 0.3.
4. The
flask was removed from the incubator and kept on ice for 20 minutes.
5. The
culture was transferred to 250ml sterile centrifuge bottle and centrifuged at
1500rpm for 15minutes at 40C
6. The
supernatant was discarded and pellet is restored.
7. Carefully
add 10ml of ice cold TSS solution and resuspend slowly.
8. Aliquot 100µl of cells in 1.5ml sterile eppendrof vials and immediately flash freeze in either liquid nitrogen or dry ice containing ethanol(Note: make sure the cells are frozen immediately else the efficiency might go down).
9. Store
at -800C IN cryobox with date of preparation and determine the transformation efficiency with any plasmid on the same day.
Preparing
the competent cells:
Reagent: TSS (transformation and
storage solution for chemical transformation)
85% LB medium
10% PEG (wt/vol, MW 8000)
5% DMSO (vol/vol)
50 mM MgCl2 (pH 6.5)
Autoclave or filters sterilize.
Store at 40C for <2 weeks.
1. Streak
the cell stock on a LB plate (added antibiotic if cells have antibiotic
resistant). Incubate the plate at 370C overnight.
2. Pick
a single, well isolated colony and inoculate it into 5ml of LB broth (plus
antibiotic). Incubate at 370C overnight with shaking at 220rpm.
3. Transfer
1ml of the standard overnight culture to a sterile 500ml flask containing 100ml
of LB medium (do not add antibiotic at this step). Incubate the cells at 370C
with the shaking at 220rpm, until OD600 reach 0.5; this usually takes 2.0-2.5
hr. heck the OD frequently when it gets beyond 0.2 to avoid overgrowth.
4. When
the culture reaches an OD600 of 0.5, chill the flask on the ice for 20 min and
the collect the cells by centrifugation at 1500rpm for 5min at 40C.
5. Resuspend
the cells in 10ml of ice cold TSS solution. Now the competent cells are ready
to be transformed.
6. Aliquot
150µl competent cells to 1.5ml tube. If they are not immediately used, cells
can be stored at 40C for maximum of 6 hr. without significant loss
of competency. The same competent cells can also be stored at -700C
for long term storage (pre-treat with liquid nitrogen or dry ice).
7. Competent
cells should give a minimum of 1x106 tranformants per µg of plasmid DNA.
8. Transformation
frequency of frozen cells is 30% of that of the fresh cells, when used within two
months.
Transforming
the cells:
1. Add
DNA (20 µl) ice cold 150µl competent cells. Thaw -700C competent
cells first by hand temperature.
(You
can transform smaller portions of cells (e.g. 75µl or 50µl), but you should
decrease the volume of DNA and media proportionately.
2. Incubate on ice for 30 min, with occasional mix.
3. Heat
shock at 420C for 2 min.
4. After
heat shock, put on ice for 2 min.
5. Add
0.8ml LB broth.
6. Shake
and incubate at 370C for 60 min.
7. Plate
(<200µl) on the appropriate agar plates which contain antibiotic.
8. Incubate
plates at 370C, overnight.
OBSERVATION:
The transformed cells were plated on medium
containing ampicillin and the negative control plate didn’t show any growth
since there were no transformed cells in that plate. The test plate showed growth indicating the
presence of transformed cells.
Negative
control shows no colonies
Test
shows few colonies
RESULT:
The organisms were transformed successfully with the
plasmid inducing resistance towards ampicillin.
Troubleshooting:
Low
efficiency of transformation was observed. It may be due to the improper
handling of the cells. The cells should be thawed and used immediately. Refreezing
of the cells can decrease efficiency. Non-optimized
tubes were used. Usage of 17 x 100-mm polypropylene tubes is the best as the
heat shock step is calibrated to the size and material of these tubes. If
different tubes are used, the heat transfer to the cells may not be optimal. An
excess DNA was used. No more that 1-10 µg of DNA should be used in 100 µl of
cells. Electroporation must have being a better method.
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