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Insertional inactivation

2014-11-26T19:50:34Z

140588572:

=== ===

 Insertional inactivation is a technique used in [[Bacterial DNA technology|bacterial DNA technology]] to identify [[Transformant|transformant]] cells which took up [[Recombinant plasmid|recombinant plasmids]]<ref>Klug,W., Cummings, M, Spencer, C., Palladino, M. (2009) Concepts of Genetics. 9th edn. San Francisco: Pearson International Edition.</ref>.Genetically engineered [[Plasmid|plasmids]] are used for this purpose because they have specific features useful for the procedure. These [[Vector|vectors]] typically carry [[Marker gene|marker genes]], which enable detection of those bacterial cells which took up the [[Plasmid|plasmid]] – whether this is the ‘native’ ([[Recircularised plasmid|recircularised]]), or the [[Recombinant plasmid|recombinant plasmid ]](one that has a fragment of foreign DNA ligated to it)<ref>Reed, R., Holmes, D., Weyers, J., Jones, A. (2013) Practical Skills in Biomolecular Sciences. 4th. Harlow, England: Pearson.</ref>. Using [[Selectable marker|selectable marker genes]] is a more specific method for selection of only those bacterial cells which took up the recombinant plasmid. Prior to [[Ligation|ligation of]] a foreign DNA insert into the plasmid, the [[Vector|vector]] is cut by [[Restriction enzyme|restriction enzyme within]] the region called a [[Polylinker|polylinker]] site<ref>Klug,W., Cummings, M, Spencer, C., Palladino, M. (2009) Concepts of Genetics. 9th edn. San Francisco: Pearson International Edition.</ref>. This is located within a gene which is used as a selectable marker. [[Ligation|Ligation of]] the DNA insert into the polylinker 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 cell|transformed bacterial cells]] carrying the [[Recombinant plasmid|recombinant plasmid]]. Figure.1.<ref>Reed, R., Holmes, D., Weyers, J., Jones, A. (2013) Practical Skills in Biomolecular Sciences. 4th. Harlow, England: Pearson.</ref> presents a range of [[Selectable marker gene|selectable marker genes]] which can be inactivated during insertional inactivation. The genes would not encode for the synthesis of their products.

 

{| width="400" border="1" cellpadding="1" cellspacing="1"
|-
|
SELECTABLE MARKER GENE 

|    PRODUCT OF THE GENE     
|-
|               lacZ/lacZ'
|         β-galactosidase
|-
|                uidA 
|         β-glucuronidase
|-
|                 lux
|            luciferase
|-
|               bla /amp
|             β-lactamase
|-
|               cat
|
         chloramphenicol                

         acetyltransferase

|-
|               gfp
|
     green fluorescent protein    

      (''Aequorea victoria)''

|}

 Figure.1. Examples of selectable marker genes and their products<ref>Reed, R., Holmes, D., Weyers, J., Jones, A. (2013) Practical Skills in Biomolecular Sciences. 4th. Harlow, England: Pearson.</ref>.

=== Example of insertional inactivation using pUC18 plasmid vector ===

[[PUC18|pUC18]] is a [[Gentically engineered plasmid|genetically engineered plasmid]] characterized by specific features. The vector contains a gene for [[Ampicillin|Ampicillin]] [[Resistance|resistance]] and an insert of the bacterial [[LacZα gene|lacZ gene]]<ref>Klug,W., Cummings, M, Spencer, C., Palladino, M. (2009) Concepts of Genetics. 9th edn. San Francisco: Pearson International Edition.</ref>. A [[Polylinker|polylinker]] site with a number of [[Restriction enzymes|restriction-enzyme sequences]] is located within the lacZ gene. The product of a functional [[LacZα gene|lacZ gene]] is an enzyme called [[Β-galactosidase|β-galactosidase]], which breaks down a chromogenic substrate called [[X-GAL|X-GAL ]]([[5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside|5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside]])<ref>Reed, R., Holmes, D., Weyers, J., Jones, A. (2013) Practical Skills in Biomolecular Sciences. 4th. Harlow, England: Pearson.</ref>. Thus, when bacterial cells containing [[PUC18|pUC18 ]][[Plasmid|plasmids]] are grown on an [[Agar medium|agar-based medium]] with added [[X-GAL|X-GAL]], the substrate is broken down. Bacterial cells containing [[PUC18 plasmid|pUC18 plasmid]] give rise to [[Blue colonies|blue colonies]]. Insertion of a fragment of foreign DNA into the [[Polylinker|polylinker]] site causes disruption of [[LacZα gene|lacZ gene]], which is no longer functional (insertional inactivation). [[Recombinant pUC18 plasmid|Recombinant pUC18 plasmids]] can be identified using [[LacZα gene|LacZα_gene]] as a selectable marker. This is done by growing bacterial cells containing the recombinant plasmid on an agar-based medium containing [[X-GAL|X-GAL]]. The disrupted [[LacZα gene|lacZ gene]] is not expressed, hence, it doesn’t code for production of [[Β-galactosidase|β-galactosidase]]. Consistently, the bacterial cells with the [[Recombinant plasmid|recombinant plasmid]] cannot break down [[X-GAL|X-GAL]], and these give rise to [[White colonies|white colonies]].

== References ==

<references />

Insertional inactivation

2014-11-26T19:20:21Z

140588572: Created page with "=== === Insertional inactivation is a technique used in bacterial DNA technology to identify transformant cells which too..."

=== ===

 Insertional inactivation is a technique used in [[bacterial DNA technology|bacterial DNA technology]] to identify [[transformant|transformant]] cells which took up [[recombinant plasmid|recombinant plasmids]]<ref>Klug,W., Cummings, M, Spencer, C., Palladino, M. (2009) Concepts of Genetics. 9th edn. San Francisco: Pearson International Edition.</ref>.Genetically engineered [[plasmid|plasmids ]]are used for this purpose because they have specific features useful for the procedure. These [[vector|vectors]] typically carry [[marker gene|marker genes]], which enable detection of those bacterial cells which took up the [[plasmid|plasmid]] – whether this is the ‘native’ ([[recircularised plasmid|recircularised]]), or the [[recombinant plasmid|recombinant plasmid ]](one that has a fragment of foreign DNA ligated to it)<ref>Reed, R., Holmes, D., Weyers, J., Jones, A. (2013) Practical Skills in Biomolecular Sciences. 4th. Harlow, England: Pearson.</ref>. Using [[selectable marker|selectable marker genes]] is a more specific method for selection of only those bacterial cells which took up the recombinant plasmid. Prior to [[ligation|ligation ]]of a foreign DNA insert into the plasmid, the [[vector|vector]] is cut by [[restriction enzyme|restriction enzyme ]]within the region called a [[polylinker|polylinker]] site<ref>Klug,W., Cummings, M, Spencer, C., Palladino, M. (2009) Concepts of Genetics. 9th edn. San Francisco: Pearson International Edition.</ref>. This is located within a gene which is used as a selectable marker. [[ligation|Ligation ]]of the DNA insert into the polylinker 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 cell|transformed bacterial cells]] carrying the [[recombinant plasmid|recombinant plasmid]]. Figure.1.<ref>Reed, R., Holmes, D., Weyers, J., Jones, A. (2013) Practical Skills in Biomolecular Sciences. 4th. Harlow, England: Pearson.</ref> presents a range of [[selectable marker gene|selectable marker genes]] which can be inactivated during insertional inactivation. The genes would not encode for the synthesis of their products.

 

{| width="400" border="1" cellpadding="1" cellspacing="1"
|-
|
SELECTABLE MARKER GENE 

|    PRODUCT OF THE GENE     
|-
|               lacZ/lacZ'
|         β-galactosidase
|-
|                uidA 
|         β-glucuronidase
|-
|                 lux
|            luciferase
|-
|               bla /amp
|             β-lactamase
|-
|               cat
|
         chloramphenicol                

         acetyltransferase

|-
|               gfp
|
     green fluorescent protein    

      (''Aequorea victoria)''

|}

 Figure.1. Examples of selectable marker genes and their products<ref>Reed, R., Holmes, D., Weyers, J., Jones, A. (2013) Practical Skills in Biomolecular Sciences. 4th. Harlow, England: Pearson.</ref>.

=== Example of insertional inactivation using pUC18 plasmid vector ===

[[pUC18|pUC18 ]]is a [[gentically engineered plasmid|genetically engineered plasmid]] characterized by specific features. The vector contains a gene for [[Ampicillin|Ampicillin]] [[resistance|resistance]] and an insert of the bacterial [[LacZα_gene|lacZ gene]]<ref>Klug,W., Cummings, M, Spencer, C., Palladino, M. (2009) Concepts of Genetics. 9th edn. San Francisco: Pearson International Edition.</ref>. A [[polylinker|polylinker]] site with a number of [[Restriction_enzymes|restriction-enzyme sequences]] is located within the lacZ gene. The product of a functional [[LacZα_gene|lacZ gene]] is an enzyme called [[Β-galactosidase|β-galactosidase]], which breaks down a chromogenic substrate called [[X-GAL|X-GAL ]]([[5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside |5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside]])<ref>Reed, R., Holmes, D., Weyers, J., Jones, A. (2013) Practical Skills in Biomolecular Sciences. 4th. Harlow, England: Pearson.</ref>. Thus, when bacterial cells containing [[pUC18|pUC18 ]][[Plasmid|plasmids]] are grown on an [[agar medium|agar-based medium ]]with added [[X-GAL|X-GAL]], the substrate is broken down. Bacterial cells containing [[pUC18 plasmid|pUC18 plasmid]] give rise to [[blue colonies|blue colonies]]. Insertion of a fragment of foreign DNA into the [[polylinker|polylinker]] site causes disruption of [[LacZα_gene|lacZ gene]], which is no longer functional (insertional inactivation). [[recombinant pUC18 plasmid|Recombinant pUC18 plasmids]] can be identified using [[LacZα_gene|LacZα_gene]] as a selectable marker. This is done by growing bacterial cells containing the recombinant plasmid on an agar-based medium containing [[X-GAL|X-GAL]]. The disrupted [[LacZα_gene|lacZ gene]] is not expressed, hence, it doesn’t code for production of [[Β-galactosidase|β-galactosidase]]. Consistently, the bacterial cells with the [[recombinant plasmid|recombinant plasmid]] cannot break down [[X-GAL|X-GAL]], and these give rise to [[white colonies|white colonies]].

== References: ==

 1. Klug,W., Cummings, M, Spencer, C., Palladino, M. (2009) Concepts of Genetics. 9th edn. San Francisco: Pearson International Edition. 2. Reed, R., Holmes, D., Weyers, J., Jones, A. (2013) Practical Skills in Biomolecular Sciences. 4th. Harlow, England: Pearson.