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Insertional Inactivation

Insertional inactivation is a technique in bacterial DNA technology that identifies transformant cells with recombinant plasmids by disrupting selectable marker genes. This process allows for the selection of bacterial cells that have taken up the recombinant plasmid, as exemplified by the loss of tetracycline resistance when a DNA insert is ligated into the tetr gene. Additionally, the blue-white selection method utilizes the inactivation of the lac-Z gene to differentiate between colonies with and without the DNA insert, aiding in the identification of successful transformants.

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27 views1 page

Insertional Inactivation

Insertional inactivation is a technique in bacterial DNA technology that identifies transformant cells with recombinant plasmids by disrupting selectable marker genes. This process allows for the selection of bacterial cells that have taken up the recombinant plasmid, as exemplified by the loss of tetracycline resistance when a DNA insert is ligated into the tetr gene. Additionally, the blue-white selection method utilizes the inactivation of the lac-Z gene to differentiate between colonies with and without the DNA insert, aiding in the identification of successful transformants.

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keeratgurdeep99
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© All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Insertional inactivation

Insertional inactivation is a technique used in bacterial DNA technology to identify transformant


cells which took up recombinant plasmids
Genetically engineered plasmids are used for this purpose because they have specific features
useful for the procedure.
These vectors typically carry marker genes, which enable detection of those bacterial cells which
took up the plasmid – whether this is the ‘native’ (recircularised), or the recombinant plasmid
(one that has a fragment of foreign DNA ligated to it)
Using selectable marker genes is a more specific method for selection of only those bacterial
cells which took up the recombinant plasmid.
Prior to ligation of a foreign DNA insert into the plasmid, the vector is cut by restriction enzyme
within the region, located within a gene which is used as a selectable marker.
Ligation of the DNA insert into the site consistently causes disruption of the selectable marker
gene – the process known as insertional inactivation.
The gene is no longer expressed, and can therefore serve for selection of transformed bacterial
cells carrying the recombinant plasmid.
Examples:
Insertational inactivation of tetR
Plasmid pBR322 contains two antibiotic resistance genes, one for ampicillin (ampr gene), and
the other for tetracycline (tetr gene). If the target DNA is inserted into tetr gene using Bam HI,
the property of resistance to tetracycline will be lost. Such recombinants would be test sensitive.

When such recombinants (containing target DNA in tetr gene) are grown into medium containing
tetracycline, they will not grow because their tetr gene has been inactivated. But they are
resistant to ampicillin because ampr gene is functional.
BLUE-WHITE SELECTION METHOD:

Another powerful method of screening for the presence of recombinant plasmid is referred to as
blue-white selection.

This method is based upon the insertional inactivation of the lac-Z gene present on the vector
(e.g. PUC19).

This gene expresses the enzyme β- galactosidase whose activity can cleave a colourless substrate
called X-gal into blue colored product.

If the lac-Z gene is inactivated due to the presence of the insert, then the enzymes is not
expresses. Hence, if after a transformation experiment the [Link] host cells are plated on an
ampicillin and X-gal containing solid media plate then colonies which appears blue are those
which have transformed cells (antibiotic resistant) but do not have the insert (express active
enzyme).

Colonies which appear white are both ampicillin resistant and have the insert rDNA and thus are
the cells to be used for future experiment.

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