Difference between revisions of "Part:BBa K831009"
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=='''Functional characterization'''==
 
=='''Functional characterization'''==
  
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As HipB neutralizes the induction of persistency caused by HipA7 in this strain, we induced our strain with IPTG at different concentrations and evaluate the persisters frequencies implementing an original protocol for persister cells isolation based on lysis (S. Cañas et al., Manuscript in preparation). For
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As HipB neutralizes the induction of persistency caused by HipA7 in this strain, we induced our strain with IPTG at different concentrations and evaluated the persisters frequencies implementing an original protocol for persister cells isolation based on lysis (S. Cañas et al., Manuscript in preparation).
  
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[[Image:BBa_K831009_1.png|center|550 px]]
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[[Image:BBa_K831009_hipB.png|center|550 px]]
  
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For this experiment with used the ''Escherichia coli'' K12 TH1269 strain (''hipA7'') which is a high persistence mutant. We evaluated the persistence frequencies of untransformed TH1269 strain and ''Escherichia coli'' K12 MG1655 (wild type) as a control. This experiment was made in LB medium supplemented with Ampicillin (100 ug/mL) for insuring plasmid maintenance.
+
For this experiment we used the ''Escherichia coli'' K12 TH1269 strain (''hipA7'') which is a high persistence mutant. We evaluated the persistence frequencies of untransformed TH1269 strain and ''E. coli'' MG1655 (wild type) as controls. This experiment was made in LB medium, supplemented with Ampicillin (100 ug/mL) for ensuring plasmid maintenance, when cultures reached stationary growth phase (ON). Bacterial cultures were incubated at 37 C and 200 rpm.
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As seen, our part functions as expected. Due to the leakage of lactose promoter, HipB antitoxin is exogenously expressed even without IPTG induction, therefor the presence of BBa_K831009 part along can reduce persistence frequencies in ''Escherichia coli'' strains.
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=='''References'''==
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1.Balaban N., et al. Bacterial persistence as a phenotypic switch. 2004. Science 305:1622.
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2.Lewis K. Persister cells. Annu Rev Microbiol. 2010;64:357-372.
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3.Maisonneuve, E,. Shakespeare, L J,.Jørgensen, M G,.Gerdes, K,. Bacterial persistence by RNA endonucleases. 2011.Proceedings of theNationalAcademy of Sciences. 10.1073/pnas.1100186108

Latest revision as of 08:07, 31 October 2012

Inducible HipB antitoxin under the control of lac promoter

HipB is the antitoxin of the HipAB toxin-antitoxin (TA) module. HipB neutralizes the toxin effect of HipA due to protein-protein interactions.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 223
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Functional characterization

As HipB neutralizes the induction of persistency caused by HipA7 in this strain, we induced our strain with IPTG at different concentrations and evaluated the persisters frequencies implementing an original protocol for persister cells isolation based on lysis (S. Cañas et al., Manuscript in preparation).

BBa K831009 hipB.png

For this experiment we used the Escherichia coli K12 TH1269 strain (hipA7) which is a high persistence mutant. We evaluated the persistence frequencies of untransformed TH1269 strain and E. coli MG1655 (wild type) as controls. This experiment was made in LB medium, supplemented with Ampicillin (100 ug/mL) for ensuring plasmid maintenance, when cultures reached stationary growth phase (ON). Bacterial cultures were incubated at 37 C and 200 rpm.

As seen, our part functions as expected. Due to the leakage of lactose promoter, HipB antitoxin is exogenously expressed even without IPTG induction, therefor the presence of BBa_K831009 part along can reduce persistence frequencies in Escherichia coli strains.

References

1.Balaban N., et al. Bacterial persistence as a phenotypic switch. 2004. Science 305:1622.

2.Lewis K. Persister cells. Annu Rev Microbiol. 2010;64:357-372.

3.Maisonneuve, E,. Shakespeare, L J,.Jørgensen, M G,.Gerdes, K,. Bacterial persistence by RNA endonucleases. 2011.Proceedings of theNationalAcademy of Sciences. 10.1073/pnas.1100186108