Electron transform system

1. Construction of our the azo dye microbial reduction system

On August 26, we constructed our plasmid pYYDT-Mtr by KOD PCR and seamless cloning master mix kit.

Figure 1. Plasmid

On August 27, the plasmid was transformed into ，E.coli WM3064 by electrotransformation and cultured
On August 28, plasmid extraction was carried out after the cultured strains were kept.
On August 29, the sensitive state of S. oneidensis MR-1 was prepared by sucrose method and transformed by electric transformation method, and then coated on solid medium.
On August 30, a single colony of S. oneidensis MR-1 was selected on solid medium for colony PCR to verify whether the transformation was successful, and the verified single colony was kept for further culture and DNA sequencing (the sequencing result is positive and shown in the result part.

Figure 2. Gene Circuit of Mtr

2. Verification of electronic production ability by MFC (microbial fuel cell)

After the transformation of S. oneidensis MR-1, we compared the electronic production ability of S. oneidensis MR-1 transformed with our target plasmid pYYDT-MTR with that transformed with empty plasmid pYYDT through MFC.
After two kinds of bacteria were cultured in parallel, they were added to two MFC-devices equally (OD₆₀₀=0.3), and the voltage difference between positive and negative electrodes of MFC was recorded every ten minutes.

Figure 3. MFC

The experimental records and results clearly show that the production capacity of our engineered strain is significantly higher than that of the empty plasmid strain.

3. Reduction of methyl orange (MO) Experiment

To testify the efficiency of our electron transfer system to break azo bond, we designed the experiment of the reduction of methyl orange (MO).
MO is a model dye for the azo dyes which is widely used in the laboratory, which bears the functional group diazenyl R−N=N−R′. And the MO shows its typical orange color due to the diazenyl R−N=N−R′ structure, and MO discolors into transparent if the azo bond is reduced. Thus, if our engineered strain shows a better decolorizing ability, that the effect of our electron transfer system can be proved clearly.

Figure 4. Mechanism of azo bond reduction

We added MO to anaerobic mineral salt medium (without electron acceptor) to 50mg/L. Then, we injected engineered S. oneidensis MR-1 (with pYYDT-MTR) into sample 1~3 and wild type S. oneidensis MR-1 (with empty PYYDT plasmid) into sample 4~6 (OD₆₀₀=0.1). In the beginning, the MO concentrations of six samples are the same. After that, we incubated these samples at 30℃ and measured the concentration of MO (measured under OD₄₆₅) per 30 minutes.

Figure 5. Samples after incubation of about 5 hours.

The result is pretty good. From Figure 5., we can see that, qualitatively, our engineered strain with the electron transfer system is much stronger than wild type. And then, we quantitatively analyzed how much we improved the reduction ability of S. oneidensis MR-1. We made the exponential fitting curve of MO concentration (Figure 6.): WT y=49.44e^-0.0014x; Exp. y=49.64e^-0.0136x. (the data analyzing process is explained in the result part).

The efficiency of our engineered stain to degrade MO is 10 times higher!

Figure 6. Quantitatively analysis (a) and (b)titled.

4.verification of our quorum-sensing system

In order to verify the function of our quorum-sensing system, we have done the experiment below. We used GFP to test our QS system. We set the modified S. oneidensis MR-1 (with pYYDT-GFP plasmid) and wild type S. oneidensis MR-1 (with empty pYYDT plasmid) into two groups. We controlled the initial concentration of each group at OD₆₀₀ at 0.05 in the 2×YT liquid culture medium and then cultured them in the Microplate Reader for 10 hours. Every 20 minutes, we measured the concentration and the green fluorescence intensity of each group. And we got the result below (the data analyzing process is explained in the result part).

Figure 7. Demonstration of QS

(Picture (a) shows the bacteria concentration change of experimental group with time. Picture (b) shows the relative GFP intensity change of experimental group with time. Picture (c) shows the relationship between the concentration of bacteria and the relative expression efficiency of GFP)
Comparing the picture(a) and the picture(b), we can see that the concentration keeps rising from the beginning. However, the GFP intensity keeps in the same value for about 100 minutes at the beginning. After 100 minutes, the GFP intensity starts to rise. On the picture(c), we can see that when the concentration is low (OD₆₀₀ lower than about 0.2), the relative expression efficiency of GFP is 0. And when the concentration is higher than a certain value (OD₆₀₀>0.2), the relative expression efficiency of GFP starts to rise until the concentration reach another certain value (OD₆₀₀=0.5). Therefore, we can draw a conclusion that our QS system does work.

Aromatic amines resistance system(aNAT)

1.Construction of aNAT expression vector

We construct the plasmid with Primestar PCR and seamless cloning master mix kit. The original aNAT gene is synthesized by GENscript.

Figure 8

2.Convenient methods of bacterial degradation

It is reported that aromatic amine puts restrain on growth of Shewanella oneidensis and ability of producing electron. The mechanism of this inhibition is that aromatic amine competes with para aminobenzoic acid, PABA for short, the precursor of a coenzyme assisting the synthesis of purine and pyrimidine in bacteria. But PABA is rich in yeast extract, which demands specific medium without yeast extract. Aerobic mineral salt medium is ideal for this experiment.

To test and verify if our new part, aNAT, can help resist this restrain, we take S.oneidensis MR-1 with plasmid pYYDT carrying aNAT as sample group, S.oneidensis MR-1 wild type and S.oneidensis MR-1 with empty pYYDT as controlling group. Sulfanilamide is selected as aromatic amine. Adding 40ug/mL sulfanilamide, the OD₆₀₀ graph significantly demonstrates aNAT lifting the inhibition of sulfanilamide.

Figure 8

Reference

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[4] Hartshorne, R. S., Reardon, C. L., Ross, D., Nuester, J., Clarke, T. A., Gates, A. J., Mills, P. C., Fredrickson, J. K., Zachara, J. M., Shi, L., Beliaev, A. S., Marshall, M. J., Tien, M., Brantley, S., Butt, J. N., and Richardson, D. J. (2009). Characterization of an electron conduit between bacteria and the extracellular environment. Proc. Natl. Acad. Sci. U.S.A. 106, 22169–22174.

[5] Cocaign A, Bui LC, Silar P, et al. Biotransformation of Trichoderma spp. and their tolerance to aromatic amines, a major class of pollutants. Appl Environ Microbiol. 2013;79(15):4719–4726. doi:10.1128/AEM.00989-13

[6] https://2018.igem.org/Team:Unesp_Brazil/Design

[7] KAWANO, F. et al. Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins. Nat. Commun. v.6 n.6256, 2015.

[8] Fernando E , Keshavarz T , Kyazze G . Enhanced bio-decolourisation of acid orange 7 by Shewanella oneidensis through co-metabolism in a microbial fuel cell[J]. International Biodeterioration & Biodegradation, 2012, 72(none):1-9.

Team:USTC/Experiment