Difference between revisions of "Featured Parts:Cell Death"

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[[Image:400px-Skull and crossbones.png|thumb|ccdB: Natural selection--now in gene form!]]
 
[[Image:400px-Skull and crossbones.png|thumb|ccdB: Natural selection--now in gene form!]]
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==The Parts==
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==The Part==
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[[Part:BBa_P1010|BBa_P1010]]
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[[Part:BBa_P1010|BBa_P1010]] The part containing the ccdB ("Death") gene.  [https://parts.igem.org/cgi/partsdb/dna.cgi?part_name=BBa_P1010 Comes in various plasmids] with different resistance (amp, amp/kan, etc) flavors.
 
<hr>
 
<hr>
  
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==Suggested Uses for these Biobricks==
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==Suggested Use==
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Use plasmids with ccdB to stick your Biobricks in!
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Use plasmids with ccdB to change your construct's host [[Help:Plasmids|plasmid vector]]
  
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==Experimental and Technical==
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==How to Use this Part==
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Once you've transformed your plasmids into a cell, it's hard to figure out which clones have actually been successful in acquiring the new DNA without checking invidividual colonies. <br>
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While there were a few [[Help:Plasmids/Construction_Plasmids#Plasmid_(backbone)|plasmid (backbones)]] shipped out, it's true we haven't included many bare plasmids.  However, some of the plasmids which we HAVE included instead are specialized [[Help:Plasmids/Construction_Plasmids#Construction_Plasmid|construction plasmids]] designed to help you clone constructs into them in the following way:
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To aid you in selecting for these successful clones, we've included three variants of the [[Part:BBa_P1010|P1010]] biobrick each of which features both the Amp, Kan, and Amp/Kan resistant plasmids as well as the ccdB gene.  This gene is currently housed between the biobrick restriction sites within a cell host (DB3.1) which tolerates the gene's presence.  However, should this plasmid containing the ccdB gene be transformed into a cell type of a different strain (ie. DH10B), the gene would cause cell death. <br>
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Thus you could take the [[Part:BBa_P1010|P1010]] cells from the last shipment, purify the plasmids restrict at the biobrick restriction sites, ligate in your gene, transform into a DH10B cell line, and the successful cells will have the ccdB gene cut out, and thus will live.  On the other hand, all of the cells which were never restricted to begin with or which re-ligated with the ccdB inserts, will die within the DH10B cell line after transformation.  Thus you will have only successful clones surviving on your plate!<br>
+
−
Since you will be restricting out the ccdB gene in order to insert your new gene of interest, the ccdB gene will no longer be part of the plasmid, and you won't have to worry about its presence interfering with any of your future experiments.
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==More Information/References==
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*Once you've transformed your plasmids into a cell, it's hard to figure out which clones have actually been successful in acquiring the new DNA without checking invidividual colonies.
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*Publications
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*To aid you in selecting for these successful clones, we've included three variants of the [[Part:BBa_P1010| BBa_P1010]] biobricks each of which features both the Amp, Kan, and Amp/Kan resistant plasmids as well as the ccdB gene. This gene is currently housed between the biobrick restriction sites within a cell host([[Part:BBa_V1005|DB3.1]]) which tolerates the gene's presence. However, should this plasmid containing the ccdB gene be transformed into a cell type of a different strain (ie. DH10B), the gene would cause cell death.
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**Bahassi, et al. 1999 J. Biol. Chem. 274: 10936-10944
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*Thus you could take the [[Part:BBa_P1010| BBa_P1010]] cells from the last shipment and
 +
*#purify the plasmids
 +
*#restrict at the biobrick restriction sites
 +
*#ligate in your gene
 +
*#transform into a DH10B cell line...
 +
*#and the successful cells will have the ccdB gene cut out, and thus will live.  On the other hand, all of the cells which were never restricted to begin with or which re-ligated with the ccdB inserts, will die within the DH10B cell line after transformation...<br>
 +
<br>
 +
Thus you will have only successful clones surviving on your plate!
 +
Since you will be restricting out the ccdB gene in order to insert your new gene of interest, the ccdB gene will no longer be part of the plasmid, and you won't have to worry about its presence interfering with any of your future experiments!
 +
 
 +
==References==
 +
*Bernard P, Gabant P, Bahassi EM, Couturier M, Université Libre de Bruxelles. <i>Positive-selection vectors using the F plasmid </i>ccdB<i> killer gene.</i>" Gene 1994 Oct 11;148(1):71-4. pmid:7926841. [http://www.ncbi.nlm.nih.gov/pubmed/7926841 Pubmed]
 +
*Bernard P, Gabant P, Université Libre de Bruxelles. <i>Cloning and/or sequencing vector</i> US Patent Number 5,910,438, 1999. [http://www.google.com/patents?vid=USPAT5910438 Google Patents]
 +
*Bernard P, Gabant P, Université Libre de Bruxelles. <i>Cloning and/or sequencing vector</i> US Patent Number 6,180,407 B1, 2001. [http://www.google.com/patents?vid=USPAT6180407 Google Patents]
 +
*Bernard P, Gabant P, Université Libre de Bruxelles. <i>Cloning and/or sequencing vector</i> US Patent Number 7,176,029 B2, 2007. [http://www.google.com/patents?vid=USPAT7176029 Google Patents]
 +
 
 +
* Bahassi, et al., Université Libre de Bruxelles. <i>Interactions of CcdB with DNA gyrase. Inactivation of Gyra, poisoning of the gyrase-DNA complex, and the antidote action of CcdA.</i>, J. Biol. Chem. 1999 274: 10936-10944. pmid:10196173. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10196173&query_hl=2&itool=pubmed_ExternalLink Pubmed]
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*[[Help:Plasmids]]
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*[[Part:BBa_P1010]]

Latest revision as of 17:53, 16 March 2010

ccdB: Natural selection--now in gene form!

The Part

BBa_P1010 The part containing the ccdB ("Death") gene. Comes in various plasmids with different resistance (amp, amp/kan, etc) flavors.

Suggested Use

Use plasmids with ccdB to change your construct's host plasmid vector

How to Use this Part

While there were a few plasmid (backbones) shipped out, it's true we haven't included many bare plasmids. However, some of the plasmids which we HAVE included instead are specialized construction plasmids designed to help you clone constructs into them in the following way:

  • Once you've transformed your plasmids into a cell, it's hard to figure out which clones have actually been successful in acquiring the new DNA without checking invidividual colonies.
  • To aid you in selecting for these successful clones, we've included three variants of the BBa_P1010 biobricks each of which features both the Amp, Kan, and Amp/Kan resistant plasmids as well as the ccdB gene. This gene is currently housed between the biobrick restriction sites within a cell host(DB3.1) which tolerates the gene's presence. However, should this plasmid containing the ccdB gene be transformed into a cell type of a different strain (ie. DH10B), the gene would cause cell death.
  • Thus you could take the BBa_P1010 cells from the last shipment and
    1. purify the plasmids
    2. restrict at the biobrick restriction sites
    3. ligate in your gene
    4. transform into a DH10B cell line...
    5. and the successful cells will have the ccdB gene cut out, and thus will live. On the other hand, all of the cells which were never restricted to begin with or which re-ligated with the ccdB inserts, will die within the DH10B cell line after transformation...


Thus you will have only successful clones surviving on your plate! Since you will be restricting out the ccdB gene in order to insert your new gene of interest, the ccdB gene will no longer be part of the plasmid, and you won't have to worry about its presence interfering with any of your future experiments!

References

  • Bernard P, Gabant P, Bahassi EM, Couturier M, Université Libre de Bruxelles. Positive-selection vectors using the F plasmid ccdB killer gene." Gene 1994 Oct 11;148(1):71-4. pmid:7926841. [http://www.ncbi.nlm.nih.gov/pubmed/7926841 Pubmed]
  • Bernard P, Gabant P, Université Libre de Bruxelles. Cloning and/or sequencing vector US Patent Number 5,910,438, 1999. [http://www.google.com/patents?vid=USPAT5910438 Google Patents]
  • Bernard P, Gabant P, Université Libre de Bruxelles. Cloning and/or sequencing vector US Patent Number 6,180,407 B1, 2001. [http://www.google.com/patents?vid=USPAT6180407 Google Patents]
  • Bernard P, Gabant P, Université Libre de Bruxelles. Cloning and/or sequencing vector US Patent Number 7,176,029 B2, 2007. [http://www.google.com/patents?vid=USPAT7176029 Google Patents]
  • Bahassi, et al., Université Libre de Bruxelles. Interactions of CcdB with DNA gyrase. Inactivation of Gyra, poisoning of the gyrase-DNA complex, and the antidote action of CcdA., J. Biol. Chem. 1999 274: 10936-10944. pmid:10196173. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10196173&query_hl=2&itool=pubmed_ExternalLink Pubmed]
  • Help:Plasmids
  • Part:BBa_P1010