Difference between revisions of "Part:BBa K831015"

Revision as of 08:01, 31 October 2012

Inducible HipA7 toxin under the control of prm promoter

HipA7 is a variant of HipA toxin of the HipAB toxin-antitoxin (TA) module. Two point mutations make this toxin responsible of the high persistence phenotype of the Escherichia coli K2 TH1269 strain.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Functional characterization

As HipA7 induces persisters formation, 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).

In this biobrick, hipA7 gene is under prm promoter (induced by CI), so we constructed inducible CI under the control of the lac promoter in order to analyze the effect of the induction of HipA7 toxin in the persisters frequency on a wild type E. coli K12 strain using IPTG.

For this experiment we used the Escherichia coli K12 MG1655 which is a wild type strain. We evaluated the persistence frequencies of untransformed MG1655 strain and E. coli TH1269 (hipA7) as controls. This experiment was made in LB medium, supplemented with Ampicillin (100 ug/mL)and Cloramphenicol 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, HipA7 toxin is exogenously expressed even without IPTG induction, therefor the presence of BBa_K831015 part along can increase 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

@@ Line 25: / Line 25: @@
 In this biobrick, ''hipA7'' gene is under prm promoter (induced by CI), so we constructed inducible CI under the control of the lac promoter in order to analyze the effect of the induction of HipA7 toxin in the persisters frequency on a wild type E. coli K12 strain using IPTG.
 [[Image:BBa_K831015.png|center|550 px]]
-For this experiment we used the ''Escherichia coli'' K12 MG1655(''hipA7'') which is a wild type strain mutant. We evaluated the persistence frequencies of untransformed MG1655 strain and ''E. coli'' TH1269 (''hipA7'') as controls. This experiment was made in LB medium, supplemented with Ampicillin (100 ug/mL)and Cloramphenicol 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 MG1655 which is a wild type strain. We evaluated the persistence frequencies of untransformed MG1655 strain and ''E. coli'' TH1269 (''hipA7'') as controls. This experiment was made in LB medium, supplemented with Ampicillin (100 ug/mL)and Cloramphenicol 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, HipA7 toxin is exogenously expressed even without IPTG induction, therefor the presence of BBa_K831015 part along can increase persistence frequencies in ''Escherichia coli'' strains.