- Human Practices
For the Contribution, we completed the experimental characterization of the previous parts (BBa_K1096002 and BBa_Q04510) and added the data of them to the corresponding BioBricks.
|BioBricks||Codes in the lab||Quantitative Characterization|
|BBa_K1096002||MazF||colony forming unit (CFU)|
|BBa_Q04510||Inverter||fluorescence / OD600|
All of these may be helpful to other teams. We hope it will make some contribution to the iGEM community.
MazF is toxin protein in MazF-MazE, toxin-antitoxin module. It was first registered in 2013 and used as an mRNA endonuclease. It kills bacteria without cracking them, which will directly influence the value of OD600.
In order to quantify the toxicity of mazF, colony forming unit (CFU) cell viability assays were used to measure functionality of the circuit(1).
Because direct expression of MazF will kill bacteria, we construct the circuit “PBad/araC-RBS-MazF-terminator-pSB1C3” and “PBad/araC-RBS-EYFP-terminator-pSB1C3” in E. coli BL21(DE3) to characterize its function.
Fig. 1 genetic circuit of PBad/araC-RBS-MazF-terminator
When 0.2% arabinose was added, the experimental group showed a sharp decrease on the CFU, which indicates the protein encoded by MazF is fatal to E. coli. It further demonstrated that the MazF encoded by J23100-RBS was able to work as a toxin.
Fig. 2 The results of CFU.The round dot indicates the non induction group, and the square dot indicates the induction group.
Inverter is mainly composed of cI repressor from E. coli phage lambda and pR promoter which is inhibited by cI repressor. It can reverse the inducer action of the upstream promoter. It was registered in 2003.
Fig. 3 Genetic circuits of Inverter
We constructed “PBad/araC-RBS-EYFP-pSB1C3” and “PBad/araC-Inverter-RBS-EYFP-pSB1C3” in E. coli BL21（DE3）to characterize its function.
For the latter circuit, cI repressor will not express in the absence of arabinose so that the fluorescence of EYFP can be observed. At the certain concentration of arabinose, PBad/araC can be activated to express cI repressor to inhibit the pR promoter，so the fluorescence of EYFP decreases.
The fluorescence/OD600 values of these two circuits were measured with or without arabinose and compared with “PBad/araC-pSB1C3” in E. coli BL21(DE3). We discovered that the downstream gene of PBad/araC can be expressed in the presence of arabinose and decrease expression without arabinose. When the inverter is added to the circuit, the effect is reversed. The result proves that inverter can function correctly and reverse the effect of PBad/araC.
Fig. 4 The results of Fluorescence Intensity/OD.Yellow represents PBad/araC, green represents PBad/araC-EYFP, and purple represents PBad/araC -Inverter-EYFP.
1. C. T. Chan, J. W. Lee, D. E. Cameron, C. J. Bashor, J. J. Collins, 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat Chem Biol 12, 82-86 (2016).