Introduction to Recombinant Genetics- Biology 350

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TRANSFORMATION AND THE INTRODUCTION OF DNA INTO CELLS Since it is an uncommon event for a cell to take up a DNA from the external environment, 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 DNA ligation products.

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

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. For E. coli and other species, physical or chemical treadments 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 brief heat 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.

Introduction of phages DNA into bacterial cells Phage DNA that carries packaging sites can be coated with phage protein either in vivo or in 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.

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 the plating 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 to 10^7. 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 of the 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 efficiently that 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 colonly 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.

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 of in 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. 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.