Transformation of Host Cells(Amrita University)


Uploaded by amritavlab on 03.02.2012

Transcript:
TRANSFORMATION OF HOST CELLS Transformation process allows a bacterium
to take up genes from its surrounding environment; i.e., transformation involves the direct uptake
of fragments of DNA by a recipient cell and the acquisition of new genetic characteristics.
There are two major parameters involved in efficiently transforming of a host. The first
is the method used to induce competence for transformation. The second 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. Calcium chloride treatment is one of the best methods for the
preparation of competent cells. In the process of transformation, the competent cells are
incubated with DNA in ice. Then it is placed in a water bath at 42ºC and subsequently
plunged in ice. This process takes 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 rapid screening of successful transformants. Blue –white selection (Alpha
complementation) can be used to differentiate between transformants and non transformants.
Materials Required: Icebox, Competent cells, DNA, LB Broth, IPTG,
X-GAL, Empty Microfuge tube, Petriplate, Spreading Rod in Ethanol, Laminar Air Flow, Floater,
Water Bath, Incubator, Micropipettes, Tips. Procedure
Arrange all the required materials in the Laminar Air Flow and flame the Bunsen burner.
Insert a fresh micropipette tip into a 1000 micro liter micropipette and transfer 200
micro liter of competent cells into an empty microfuge tube taken from the ice box.
Place the tube back in the ice box. Take 10 micro liter micropipette, insert a
fresh micropipette tip into it and transfer 10 micro liter of DNA to this microfuge tube.
Store the tube in ice for 30 minutes. Now place the tube in a floater and keep it
in the water bath at 42º Celsius for 95 seconds. Take the tube from the water bath and place
it back in the ice box for 2 minutes. Now insert a fresh micropipette tip into the
1000 micro liter micropipette and transfer 800 micro liter of LB Broth from the conical
flask to the tube. Place the tube in a floater and keep it in
the water bath at 37º Celsius for 45 minutes. After 45 minutes, take the tube from the water
bath and place it back in the ice box. Switch off the light in the Laminar Air Flow.
Now take the 100 microliter micropipette, insert a fresh micropipette tip and transfer
40 microliter of X Gal into a petri plate. Take the 10 microliter micropipette, insert
a fresh micropipette tip and transfer 7 microliter of IPTG into the same petri plate.
Insert a fresh micropipette tip into the 1000 microliter micropipette and transfer 200 microliter
of solution containing competent cells, DNA and LB Broth to the same petri plate.
Now dip the spreading rod in ethanol and burn it in flame.
Allow to cool it for some time. Using this rod spread the solution in the
petri plate evenly. After spreading the solution, close the petri
plate. Ensure the process of adding and spreading
the solutions in the petri plate do not take too much time.
Observations After 12-16 hours, on removing the petri plate
from the incubator, we can see blue and white colonies in the petri plate.