Results

1. The Tolerance of Ag+

According to the general range of the concentration of \(\ce{Ag+}\) (1～40μM) obtained by the SAD model, different gradients of the solid medium are prepared. The same number of Shewanella are inoculated in each group, and the growth of it at different concentrations of \(\ce{Ag+}\) is observed (figure1). The results are more obvious when the number of inoculated Shewanella is small. From the point of view of the number of colonies, the activity of Shewanella decreases with the increase of the concentration of \(\ce{Ag+}\).

We tested the growth density of shewanella under different concentrations of \(\ce{Ag+}\) in 12 hours. We focus on concentrations of 10 μM and higher. The density of SW-MR-Atox1 is higher than that of the SW-MR-1, but when there is kanamycin, the density decreases, which means that kanamycin has some negative influence on SW-MR-Atox1.(figure2)

2. Plasmid Construction and Electrical Transformation

BpfA(C-terminal 1000bp), AgBP2+KanR(910bp), and AggC(N-terminal 1000bp) fragment is amplified from the available plasmid in our laboratory, and the PCR product is purified by agarose gel electrophoresis (figure3).

The existing plasmids are digested by EcoR1 and Kpn1, and the pUC57mini plasmid skeleton is recovered by agarose gel electrophoresis (figure4).

Three target fragments BpfA, AgBP2+KanR, and AggC are sequentially linked in pUC57mini plasmid (figure5) using multi-fragment seamless cloning technology (Novezan ClonExpress®). Then we get the target plasmid.

Then the constructed plasmid is sequenced to confirm whether it is consistent with the previous design, pUC57mini-BpfA-AgBP2-Linker-KanR-loxP-AggC [BBa_K4134078], which means that the sequence matching degree reaches 100%.

The above procedure is repeated by replacing AgBP2 with Atox1, and we get the backup plasmid, pUC57mini-BpfA-Atox1-Linker-KanR-loxP-AggC [BBa_K4134067], which ensures the electrical transformation of Shewanella carried out smoothly.

First, we introduce the plasmid into E.coli DH 5α for amplification, then we recover it by alkali lyse-adsorption column method, and cleave it by EcoRⅠ and KpnⅠ. Lastly, we purify the target DNA fragments by agarose gel electrophoresis (figure8).

In the first round of experiments, we use AgBP2 as a silver-binding protein to build the device [BBa_K4134077] and screen recombinants for ampicillin resistance. However, the electrotransformation fails many times. In several repetitions, we learn that the electrotransformation of Shewanella is relatively difficult. So, we redesign the device [BBa_K4134066], try another silver-binding protein Atox1, and continuously adjust the voltage intensity of electrotransformation. As a consequence, we finally get a qualified recombinant with kanamycin resistance (KanR) at a lower voltage (1.2kV) of electrical pulses. And AtoxⅠ, the upstream gene of KanR is proved to be displayed on the surface of S. oneidensis.

3. The Experiments for Biofilm Growth

The modified Shewanella is cultured in LB Culture without Ag+, and the OD at 590nm is measured after 24h and 48h (figure10) to reflect the biofilm thickness of adherent growth Shewanella. The results show that the biofilm thickness of the modified Shewanella was slightly higher than that of the control group.

4. Silver Nanoparticles Detection

1mM \(\ce{AgNO3}\) solution is added to the culture to detect silver nanoparticles:

• SW is the control group, it is wild-type Shewanella.

• SW-Atox1 (SA) is the experimental group, and the silver ion-binding protein Atox1 is fused and expressed in the C-terminal of membrane protein BpfA.

TEM

The results of transmission electron microscopy (TEM) (figure11(a))show that silver nanoparticles are indeed obtained on the cell surface, and compared with the wild type, the size of the recombinant nanoparticles was smaller and the density was higher.

UV-visible Spectroscopy

The characteristic absorbance of silver nanoparticles (410nm) is detected (figure12). SW-AtoxⅠ has more silver nanoparticles than the control group.

XPS (X-Ray Photoelectron Spectroscopy)

X-Ray Photoelectron Spectroscopy (XPS) can not only determine the surface elements and content but also the valence of elements, indicating that SW-AtoxⅠ has more elemental silver than the control group (figure13).

Energy Dispersive X-ray Spectroscopy (EDS)

Energy Dispersive X-ray Spectroscopy (EDS) is used to detect the composition and content of elements, indicating that SW-AtoxⅠ has higher silver content than the control group (figure14).

Determination of Silver Nanoparticles under Different Silver Concentrations (figure15).

5.Half-cell Experiment

In the half-cell experiment, we build a three-cell system and designed four working electrodes (figure16).

They are ordinary carbon paper, ordinary carbon paper + reduced graphene oxide, and further, adding chemically synthesized nano-silver particles, and bio-synthesized nano-silver particles in this experiment. The I-t curve was measured at a constant voltage of 0.2V. The electrode with improved stains obtains a higher peak current than the ordinary electrode.

In addition, comparing the chemically synthesized silver nanoparticles with the biosynthetic ones, we use 5% as much silver as is used in our reference to achieve a similar max current intensity (figure17).