Difference between revisions of "Part:BBa K831001"

 
 
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K831001 short</partinfo>
 
<partinfo>BBa_K831001 short</partinfo>
Line 19: Line 18:
 
<partinfo>BBa_K831001 parameters</partinfo>
 
<partinfo>BBa_K831001 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
 +
=='''Functional characterization'''==
 +
 +
For assessing the functionality of BBa_K831001, we built the composite part [https://parts.igem.org/Part:BBa_K831009 BBa_K831009], in which HipB antitoxin is expressed under the control of lactose promoter
 +
 +
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).
 +
 +
[[Image:BBa_K831009_1.png|center|550 px]]
 +
 +
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

Latest revision as of 04:27, 3 October 2012

HipB antitoxin from Escherichia coli K12

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 17
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Functional characterization

For assessing the functionality of BBa_K831001, we built the composite part BBa_K831009, in which HipB antitoxin is expressed under the control of lactose promoter

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 1.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