Team:FAFU-CHINA/Experiments

Module 2: Metal-trap

Module 3: Safeguard

Bacillus megaterium is a type of phosphate-solubilizing bacteria that play a significant role in phosphorus nutrition by enhancing its availability to plants through release from organic soil P pools by mineralization and is also a type of rhizosphere bacteria are known to play an essential role in the management of heavy metal stresses in plants. As our chassis, we have intended to highlight Bacillus megaterium as a metal-trap by using genetically engineering method.

MTs(metallothioneins)-are low-molecular weight, cysteine-rich metal-binding proteins found in wide variety of organisms including bacteria, fungi and all eukaryotic plant and animal species. MTs contain 61-62 amino acids and are structually composed of two globular metal-binding domains(α and β) with metal ion. The C-terminal α domain contains 11 cysteines and the N-terminal β domain contains 9 cysteines. The 20 cysteine residues in MTs allow it to bind up to a total of 7 cadmium ions.[1]Several systems have been developed to allow for stable construction and high level expression of recombinant proteins in bacteria. To avoid being degradated by proteolytic enzyme, the expression of MT in frame with GST allows for stable expression. Im previous research, the fusion protein was prone to increase metal ion sequestration in E.coli, this time we intended to try to express fusion protein in our chassis.

MBP(engineered from PbrR and MerR respectively)-PbrR and MerR are members of the MerR family of metal-sensing regulatory protein, acts a effective Pb(II) and Hg (II) capturer respectively. According to earlier research, the DNA binding domain and metal binding domain can function individually and the constructed peptide can form a stable dimer with its mercury and lead binding affinity remaining. In order to reduce side effects of over-expression, Peking University tandemed two copies of metal binding domain of MerR and PbrR.

Previous research showed that different localizations affect metal biosorption capability. [2]In this regard, MBPs were expressed in either the periplasmic and outer membrane by using DsbA and Lpp-Omp fusion partner respectively.

Reference:

[1]Haq, F., Mahoney, M., & Koropatnick, J. (2003). Signaling events for metallothionein induction, 533, 211т226. https://doi.org/10.1016/j.mrfmmm.2003.07.014

[2]Kao, W., Chiu, Y., Chang, C., & Chang, J. (2006). Localization Effect on the Metal Biosorption Capability of Recombinant Mammalian and Fish Metallothioneins in Escherichia coli, 1256т1264.

Fig 1 Toggle switch, a mechanism that is triggered if a human operator becomes incapacitated, allows cells to live as long as an operator compound (anhydrotetracycline, ATc) is present.

Biochtainment systems that couple enviromental sensing with circuit-based control of cell viability could be used to prevent escape of genetically modified microbes into the enviroment. In order to guarantee our chassis’ safety of the application in reality , we have taken a robust and significant strategy, dubbed ‘Toggle Switch’(), based on a circuit which the LacI and TetR transcription factors are reciprocally repressive.

Overview

For Human Practice, we have come up with a common question to President Shi that “If our project needs to be applied in practice, what will be the specific operation processes? And of what aspects shall we be conscious? ”. It is a essential reminder of his answers tha there is the need for constant care. We decided to customize a ‘kill switch’ as a safeguard for our project, simultaneously harbors a neutral statet that allows billions of microbes to happily thrive and avoid unexpectedly escaping of GEMs into the enviroment and transferring of non-natural plasmid DNA to existing soil bacteria. ‘Toggle Switch’, based on a circuit which the LacI and TetR transcription factors are reciprocally repressive, but in which the expression off TetR is favoured owing to modifications in the strength of the ribosommal binding sites of the two transcription factors. Inhibition of TetR expression by anhydrotetracycline(ATc), a compound that is not normally found in nature, is necessary of expression of LacI. LacI directly inhibits expression of a lethal toxin or indirectly prevents inhibition of the expression of our target genes. Removal of ATc from the enviroment activates the expression of TetR, which leads to cell death.

Reference

Haynes, K. A. (2016). Synthetic biology: Building genetic containment. Nature Publishing Group, 12(2), 55–56. https://doi.org/10.1038/nchembio.2004

Osório, J. (2015). Genetic kill switches — a matter of life or death. Nature Publishing Group, 1979(December), 2015. https://doi.org/10.1038/nrg.2015.29

Chan, C. T. Y., Lee, J. W., Cameron, D. E., Bashor, C. J., & Collins, J. J. (2015). for bacterial containment. Nature Chemical Biology, (December), 1–7. https://doi.org/10.1038/nchembio.1979