Engineering Success

Engineering success is always a very important part of both iGEM and synthetic biology. Design–Build–Test–Learn (DBTL) cycle is traditional engineering guidance for synthetic biology. Following this principle, our iGEM project carried out a series of experiments and researches to achieve engineering success.

Engineering

Design

MFC can directly convert chemical energy stored in organic matter into electricity and have important applications in power generation and wastewater treatment. However, current MFCs generally exhibit undesirably low power density. So how to improve the power density?

With this question, we interviewed an investor in the field of biotechnology, who knows a lot about electrogenic microorganisms. He told us that MFC has three main limiting factors: electrodes, device design, and strains. And for strains, the sluggish transmembrane and extracellular electron-transfer processes are the biggest flaws. After brainstorming, we decided to solve this problem with synthetic biology.

Introducing Shewanella transmembrane and outer-membrane silver nanoparticles can boost the charge-extraction efficiency, reports a study published in 2021 in Science. They added chemically synthesized AgNPs to electrodes and their MFC reached the highest current density ever. This research aroused our interest in AgNPs. We plan to display a silver-binding protein on the membrane of Shewanella to help it capture silver ions and reduce silver into nanoparticles so as to boost charge-extraction efficiency in Shewanella microbial fuel cells.

Built

In our project, silver is a double-edged sword. It can both increase the current density of bacteria and kill bacteria. We need to consider the proper silver concentration. So, we started to design our first model.

We constructed a Suppressed-Active-Dead (SAD) Model according to the research of Haque et al. This model describes the inhibition of E. coli by silver ions. This model helped us better understand our work. After demonstrating that the model was widely available, we used it to estimate the amount of silver ions in the pre-experiment and help us narrow down silver concentrations and built a concentration gradient. See more in Model

Test

We carried out wet lab experiments and proved that silver ions of 10μmol/L and below do not harm the growth of Shewanella cells. However, the results do not agree with the model at higher silver ion concentrations.

Learn

We learned that we need to model the growth curve according to the conditions under which we grow bacteria. We revised the model parameters based on experimental data to make the model more accurate.

Design

For the MFC design, we plan to add a small amount of silver nitrate to the anode chamber. We display silver-binding protein on the C-terminal of BpfA, a large membrane protein of _Shewanella_. Silver ions are captured on the membranes and then reduced _in situ_ by cellular metabolism-generated electrons into the transmembrane silver nanoparticles inside and traversing the cell membranes. Generally, electrons are transferred between Fe3+/Fe2+ redox centers through a multi-step hopping process. Since the electronic conductivity of metals is much higher than the redox center-mediated charge transfer process in cytochromes, transmembrane and outer membrane silver nanoparticles may act as metal shortcuts to bypass redox center-mediated slow electrons(Cao B, Science,2021). During the transfer process, it is in direct contact with the external electrode to extract the charge more efficiently and improve the current density of the MFC.

Built

To realize the fusion expression of silver-binding protein and Shewanella membrane protein, we built a new recombination device. It includes BpfA, the Carbon-terminal 1000bp sequence of a large membrane protein; AgBP2, a silver binding protein; Kanamycine resistance to select recombinants; AggC, the Nitrogen-terminal sequence for the downstream gene of BpfA.

When the device was transferred into Shewanella, the silver-binding protein could recombine to the Carbon-terminal of BpfA for silver in situ reductions.

Test

In the first round of experiments, we used AgBP2 as a silver-binding protein and designed our homologous recombination device, and built the plasmid to amplify the device. However, the electrotransformation in 1.8kV/mm failed a few times. The clone that can grow on a plate with kanamycin is our target recombinant but we didn’t see any of them.

Learn

From those failures, we learned that the electrotransformation of Shewanella is relatively difficult. So we sought help from our partner, XJTLU-China, and they advised us to adjust the voltage intensity of electrotransformation continuously. And our adviser encouraged us to try another silver-binding protein, Atox1(204bp), which is a larger silver-binding protein than AgBP2(36bp).

Redesign and Built

We redesigned and built our new device with Atox1, then carried out electrotransformation at 1.2 kilovolts per millimeter. Finally, we get a qualified recombinant.

Test

• First, this recombinant clone can grow on a plate with kanamycin.

• Furthermore, we carried out silver nanoparticle tests and half-cell current tests.

• The ability to produce silver nanoparticles was proved by TEM, UV-visible Spectroscopy, EDX, and XPS results.

• The improvement of MFC current density is proved by half-cell experiments in comparison.

The results of TEM show that silver nanoparticles were obtained on the cell surface. Compared with the wild type,  the nanoparticle of our strain has a smaller size and higher density. EDX and XPS results confirm those nanoparticles are silver in the correct valence.

In the half-cell experiment, we built a three-electrode system and designed four working electrodes. It's worth noting that we added chemically synthesized nano-silver particles and bio-synthesized ones as we designed. The I-t curve was measured at a constant voltage of 0.2V. The electrode with our stains obtains a higher peak current than the ordinary electrode. In addition, comparing the chemically synthesized silver nanoparticles with the biosynthetic ones (add 1mM Ag+), we use only 5% as much silver as is used in our reference to achieve a similar current intensity.

Learn

We confirm that we built the strain as we designed. The bio-synthesized silver nanoparticles could boost charge-extraction efficiency in Shewanella microbial fuel cells.

To date, we finished the three Design-Build–Test–Learn cycles, which is helpful for our final success throughout the whole program.

For detailed experiment results, you can check on Results page

References

1. Li, X.-Y., Zhou, X.-D. & Hu, J.-M. A straightforward and reliable evaluation of Ag(I) binding affinity mediated by a peptide ligand for constructing an efficient sensing platform. Talanta 226, 122089 (2021).
2. Cao, Y. et al. A Synthetic Plasmid Toolkit for Shewanella oneidensis MR-1. Front. Microbiol. 10, 410 (2019).
3. Sun, P., Li, K., Lin, K., Wei, W. & Zhao, J. Ag 2 S Quantum Dots for Use in Whole-Cell Biohybrid Catalyst for Visible-Light-Driven Photocatalytic Organic Pollutant Degradation. ACS Appl. Nano Mater. 5, 9754–9760 (2022).
4. Wang, X. et al. An unexpected all-metal aromatic tetranuclear silver cluster in human copper chaperone Atox1. Chem. Sci. 13, 7269–7275 (2022).
5. Ge, C. et al. Biotic Process Dominated the Uptake and Transformation of Ag + by Shewanella oneidensis MR-1. Environ. Sci. Technol. 56, 2366–2377 (2022).
6. Hall Sedlak, R. et al. Engineered Escherichia coli Silver-Binding Periplasmic Protein That Promotes Silver Tolerance. Appl Environ Microbiol 78, 2289–2296 (2012).
7. Lin, T. et al. Engineered Shewanella oneidensis-reduced graphene oxide biohybrid with enhanced biosynthesis and transport of flavins enabled the highest bioelectricity output in microbial fuel cells. Nano Energy 50, 639–648 (2018).
8. Bai, X. et al. Engineering synthetic microbial consortium for efficient conversion of lactate from glucose and xylose to generate electricity. Biochemical Engineering Journal 172, 108052 (2021).
9. Yang, Y. et al. Enhancing Bidirectional Electron Transfer of Shewanella oneidensis by a Synthetic Flavin Pathway. ACS Synth. Biol. 4, 815–823 (2015).
10. Song, R.-B. et al. Graphene/Fe 3 O 4 Nanocomposites as Efficient Anodes to Boost the Lifetime and Current Output of Microbial Fuel Cells. Chem. Asian J. 12, 308–313 (2017).
11. Zou, L., Huang, Y., Long, Z. & Qiao, Y. On-going applications of Shewanella species in microbial electrochemical system for bioenergy, bioremediation and biosensing. World J Microbiol Biotechnol 35, 9 (2019).
12. Zhou, G., Yuan, J. & Gao, H. Regulation of biofilm formation by BpfA, BpfD, and BpfG in Shewanella oneidensis. Front. Microbiol. 6, (2015).
13. Cao, B. et al. Silver nanoparticles boost charge-extraction efficiency in Shewanella microbial fuel cells. Science 373, 1336–1340 (2021).
14. Sivakumar, K. et al. Surface display of roGFP for monitoring redox status of extracellular microenvironments in Shewanella oneidensis biofilms. Biotechnol. Bioeng. 112, 512–520 (2015).
15. Gudipaty, S. A. & McEvoy, M. M. The histidine kinase CusS senses silver ions through direct binding by its sensor domain. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1844, 1656–1661 (2014).