Part:BBa_K4604024
piG_23 (tetR_mazF)
This BioBrick consists of the tetR repressor, the tet promoter, the modified T7 RBS, mazF and the rrnB terminator.
Biology
A bacterial toxin-antitoxin (TA) system, like MazE/F, is a genetic module that includes a pair of two genes, consisting of a stable toxin gene and an unstable antitoxin gene. Such systems exist either on extrachromosomal elements or on bacterial chromosomes. MazF serves as a toxin, functioning as an endoribonuclease, capable of cleaving both single and double stranded cellular RNA. As soon as the concentration of toxin surpassses the neutralization capacity of the antitoxin, the toxin leads to growth inhibition. The antitoxin protein MazE neutralizes the toxin by forming a protein-protein complex in the form of a heterohexamer. It consists of two mazE monomers, which are bounded with four MazF monomers. To gain insights on the functionality of the antitoxin-toxin system we performed several experiments.
Characterization
We needed to verify the functionality of the toxin MazF, for this we used a construct that contains an illegal restriction site on the tetR repressor due to time constraints. We identified the time span in which the toxin MazF kills the cells and thereby confirmed its toxicity. This was accomplished by an assay consisting of OD measurements and colony-forming-units (CFU). Cell toxicity was tested in a liquid culture with different inducer concentrations. First a western blot was performed to detect MazF expression.
Figure 1: MazF toxin expression for different inducer concentrations. Detection of recombinantly expressed, FLAG-tagged MazF toxin with SDS-PAGE followed by western blot using anti-FLAG antibody. Loading control: RNA polymerase subunit. Western blot with samples from different time points of pGGAselect in MG1655 in M9 medium, induced and uninduced in Lämmli buffer; Western blot with samples from different time points of piG_23 in MG1655 in M9 medium, induced in non-degrading buffer.
Induced expression of MazF was confirmed by western blot. The bands at 15 kD are the monomeric MazF. Since MazF also forms an unstable dimer, the faint bands at a height of 30 kD are presumably the MazF dimer. Moreover, we have observed an accumulation of MazF with increasing culture time.
To determine the toxicity of the expressed MazF, we performed a toxicity assay consisting of a growth measurement and colony-forming-unit (CFU).
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| Figure 2: Growth assay of E. coli MG1655, containing piG_23 induced with different DOX concentrations in M9 medium. OD600= 0.5 of culture samples were measured using ThermoScientific NanoDrop 2000c Spectrophotometer. | Figure 3: E. coli MG1655 colony forming units (CFU) assay. CFU/mL values for each timepoint for piG_23 |
A clear growth inhibition was observed in cultures of piG_23 (BBa_K4604024) induced with doxycycline concentration of 50 ng/ml and higher. To make sure that the toxicity is not due to the antibiotic nature of our inducer we tested different concentrations of doxycycline and their effect on growth of cells not containing mazF.
Figure 4: E. coli MG1655 growth curve comparison in LB medium over 20 hours with different DOX concentrations.DOX added after an OD 600 of 0.4 was reached. OD 600 measurement of culture samples using ThermoScientific NanoDrop 2000c Spectrophotometer.
After this we came to the conclusion that concentrations of up to 100 ng/mL do not inhibit cell growth. These results clearly prove the toxic effect MazF has on cells and their proliferation.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 701
Illegal XbaI site found at 616 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 701
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 701
Illegal BglII site found at 710
Illegal BamHI site found at 1105
Illegal XhoI site found at 1114 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 701
Illegal XbaI site found at 616 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 701
Illegal XbaI site found at 616 - 1000COMPATIBLE WITH RFC[1000]
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