Difference between revisions of "Part:BBa K831009"
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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). | 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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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. | 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. | ||
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 223
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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).
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