BT 403+Mod III+NKJ+Lecture 3 Transformation+and+the+Introduction+of+DNA+Into+Cells 2011

7/28/2019 BT 403+Mod III+NKJ+Lecture 3 Transformation+and+the+Introduction+of+DNA+Into+Cells 2011

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BT-403 MOD-III NKJ LECTURE: TRANSFORMATION AND THE

INTRODUCTION OF DNA INTO CELLS

Since it is an uncommon event for a cell to take up a DNA from the externalenvironment, it is rare that it will take up two DNA molecules. Therefore, introduction of

DNA into cells is like a purification step that allows the separation of mixture of DNAligation products.

Cloning can also provide a large amount of purified recombinant DNA as cultures aregrown from well separated colonies.

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Introduction of Plasmids into Bacterial Cells

In nature the uptake of DNA from the media is limited to a few genera (Bacillus

and Streptococcus) that have mechanisms for binding and uptake. ForE. coli and otherspecies, physical or chemical treatments are needed to enhance DNA uptake. These

treatments make the cells "competent" to take up DNA and cells that undergo such

treatments are called "competent cells".There are a number of treatments that have been developed to make cells

competent. Here are a few treatments:

CaCl2 - Precipitates DNA onto surface of cell and makes membranes fragile. A briefheat shock peterbs the membrane and allows some DNA to enter into the cell.

DMSO - Makes membranes permeable.

LiCl - Like CaCl2

Electroporation - pulse used to peterb membrane and sweep media contents into cells.Requires that cells be washed and suspended in water to decrease conductivity.

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Introduction of phages DNA into ba cterial cells

Phage DNA that carries packaging sites can be coated with phage protein eitherin vivo

orin vitro

.

In vivo systems for coating, such as the ssDNA synthesis and packaging carried by the

f1 origin is efficient at making coated ssDNA which is readily infectious.

In vitro coating using packaging extracts is an efficient way to encapsulate DNA that

carries a packaging site.

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Once the DNA is packaged in the phage coat, infeciton of target cells is very efficient.

Transformation efficiency

Each of the transformation and transfection methods have associated efficiencies.Transformation efficiency is calculated by dividing the number of colonies of plaques

observed per mass of DNA used in the transformation.

For example, if 10 ng of DNA was used to transform 100 L of competent cells and

during the transformation the total volume of cells was diluted to 1 mL followed by theplating of 50 L of the mix, we would have plated an equivalent to 1/20 of the input

DNA or 0.5 ng. If 400 colonies grow on the plate, then the transformation efficiency

would be:

400 colonies / 0.5 ng DNA *1000 ng/g = 800,000 colonies/g DNA or 8 x 10^5.

Transformation efficiency for CaCl2 treated cells is typically on the order of 10^6 to10^7. Other chemical methods for preparing competent cells have resulted in higher

efficiencies for some strains of bacteria, approaching 10^9 transformants/g DNA.

Electoporation can attain transformation efficiencies up to 10^12 transformants/g DNA.

Transformation efficiencies, reported on a mass basis, will differ depending on size ofthe plasmid. Small plasmids will transform more efficiently than very large plasmids.

The purity and form of the DNA being transformed also will impact transformation

efficiency. Supercoiled DNA purified on a CsCl gradient will transform more efficientlythat a mixture of ligation products.

Selection of recombinants

Once DNA has been introduced into the cell, recombinants will need to be selected.

a) We have already discussed the blue / white colony selection for recombinant

plasmids, and the same principle applies for recombinant phages that carry an

interruptable lacZ gene except instead of colonies you get blue or white plaques.

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b) Cloning into the lambda cI gene changes plaque morphology from turbid to clear.

c) Cloning into lambda compatability elements changes the vector from incompatable

to compatable for grown in a P2 lysogenic bacterial strain.

d) Cloning fragments into lambda vectors, followed by a round ofin vitro packaging, is

a good way to select for a narrow range of fragment sizes.

Introduction of DNA into Mammalian Cells

a) Transfection of DNA into mammalian cell cultures is done by precipitation of the

DNA onto the cell surface using calcium phosphate.

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Transfection of DNA into mammalian cells is also done with PEG (polyethyleneglycol)

which causes cell fusion.

a) Microinjection of DNA depends on injecting DNA directly into the nucleus via very

tiny glass capillaries.

b) A gene gun can be used to shoot DNA coated carbon particles into cells using a

ballistic system.

Introduction of DNA into Plant Cells

Getting DNA into plant cells can be a problem because the cell wall interferes. Cell

walls can be digested using cellulases to release protoplasts. Protoplasts can then be

transfected with DNA using the methods described for mammalian cells.

Another way to introduce recombinant DNA into plant cells is to use the

Agrobacterium vector.

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BT 403+Mod III+NKJ+Lecture 3 Transformation+and+the+Introduction+of+DNA+Into+Cells 2011

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