Part:BBa_K4604019
piG_03 (tetR_RiboK12_mazF)
BioBrick piG_03 is a construct consisting of the tet promoter/repressor, a riboswitch for AdoCbl, mazF and the rrnB terminator. The backbone we used in the experiments is pGGAselect. The tet promoter is a BioBrick from iGEM Freiburg 2022 (BBa_K4229059).
Usage and Biology
Toxin-antitoxin systems (TA-systems) plays a crucial role in plasmid stability for naturally occurring plasmids [1]. Usually, the toxin targets essential cellular functions and causes growth arrest or cell death, to which the antitoxin acts as a counterpart. Toxin and antitoxin exhibit differences in their stability and lifespan [2]. While the antitoxin has a shortened lifespan due to its sensitivity to degradation, the toxin has a longer lifespan and is more stable. If the plasmid containing the TA-system is lost, the antitoxin is rapidly degraded and the toxin concentration increases, leading to cell death. Therefore, when first discovered, TA systems were called “addiction modules” that ensure plasmid retention. Riboswitches occur naturally as regulators of gene expression. They are encoded as DNA sequences which exert their regulatory effect upon transcription by folding into a complex 3D RNA structure. Generally, a riboswitch can fold in two ways: Either with the ribosome binding site (RBS) being available or unavailable for the ribosome. Upon binding to the target compound, for example AdoCbl, a configurational change in the riboswitch leads to an unavailability of the RBS. In E. coli, a riboswitch regulates the expression of the btuB gene which encodes for a corrine transporter protein BtuB. It has been shown that sufficient AdoCbl concentrations cause the riboswitch to undergo a conformational change resulting in lower BtuB expression [3]. We decided on using this riboswitch to downregulate the toxin expression when AdoCbl is produced in sufficient amounts.
Characterization
MazF was tested in combination with the riboswitch to assess successful regulation of toxin expression. For this, we conducted a toxicity assay via OD measurements and CFUs. As opposed to the assay performed with BBa_K4604024 this had to be done in M9 medium to prevent the riboswitch from being triggered by the AdoCbl concentration in LB-Medium. This caused a general decrease in growth.
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| Figure 1: Growth assay of E. coli MG1655, containing piG_03 over 24 hours in M9 medium with different DOX concentrations. OD600= 0.5 of culture samples were measured using ThermoScientific NanoDrop 2000c Spectrophotometer. | Figure 2: E. coli MG1655 colony forming units (CFU) assay. CFU/mL values for each timepoint for piG_03 and control pGGAselect. |
These graphs indicate inhibited toxicity of MazF if regulated by the riboswitch.
References
[1] Ni S, Li B, Tang K, Yao J, Wood TK, Wang P, et al. Conjugative plasmid-encoded toxin–antitoxin system PrpT/PrpA directly controls plasmid copy number. Proceedings of the National Academy of Sciences of the United States of America [Internet]. 2021 Jan 22;118(4). Available from: https://doi.org/10.1073/pnas.2011577118
[2] Brzozowska I, Zielenkiewicz U. Regulation of toxin–antitoxin systems by proteolysis. Plasmid [Internet]. 2013 Jul 1;70(1):33–41. Available from: https://doi.org/10.1016/j.plasmid.2013.01.007
[3] Nou X, Kadner RJ. Adenosylcobalamin inhibits ribosome binding to btuB RNA. Proceedings of the National Academy of Sciences of the United States of America [Internet]. 2000 Jun 13;97(13):7190–5. Available from: https://doi.org/10.1073/pnas.130013897
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 710
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 883
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1518
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