. Proof of Concept .

Anti-icing

Introduction

Based on the previous experiments, we not only secreted the two AFPs into extracellular circumstances, constructed two effective transcription systems based on the cspA CRE, improved the cold-resistance of engineered bacteria, but also realized the adsorption on crop roots of engineered bacteria. To prove that our engineered bacteria which express TmAFP and SfIBP are capable of antifreeze at our eventual implementation, TmAFP and SfIBP’s anti-icing abilities were characterized.

AFPs could prevent water from freezing

E. coli BL21(DE3) harboring BBa_K4907000_pET-28a(+) were cultivated and induced by 1 mM IPTG at 20 ℃, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by SDS-PAGE and Coomassie blue staining. As shown in the gel image (Fig. 1), the target protein (25.5 kDa) can be observed at the position around 25 kDa on the purified protein lanes (FR).

Fig. 1 SDS-PAGE gel image of SfIBP protein

E. coli BL21(DE3) harboring BBa_K4907001_pET-28a(+) were cultivated and induced by 0.7 mM IPTG at 20 ℃, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by SDS-PAGE and Coomassie blue staining. As shown in the gel image (Fig. 2), the target protein (12.2 kDa) can be observed at the position around 10 kDa on the purified protein lanes (FR).

Fig. 2 SDS-PAGE gel image TmAFP protein

SfIBP and TmAFP could decrease the freezing temperature of water

TH is the ability to lower the freezing point of water, which is defined as the gap between incomplete melting point (Th) and freezing point (To). To test the TH ability of TmAFP and SfIBP, BSA was set as the negative control. Each protein was diluted to 50 mM and then was measured by NETZSCH F3 Differential Scanning Calorimetry (please see Experiment for details).

As shown in Fig. 3a, Th was determined as -1 ℃, which is lower than the melting temperature. Then To was defined as the beginning temperature of the exothermic peak. The two AFPs, SfIBP and TmAFP, have significant TH activity compared to BSA (Fig. 3b).

Fig. 3 Characterization results of the antifreeze protein. a The DSC scanning curves of BSA. b The DSC scanning curves of SfIBP. c The DSC scanning curves of TmAFP. d The TH of three proteins.

TmAFP and SfIBP could inhibit ice recrystallization

Ice recrystallization inhibition, namely IRI, after a freeze-thaw circle, the recrystallization occurrence temperature of AFPs was also lower than that of BSA too, which indicates the high IRI activity of AFPs.

Conclusion

Our experimental results confirmed that SfIBP and TmAFP can prevent freeze. In our eventual implementation, engineered bacteria will secret AFPs into the soil to protect crops. Hence, further characterization should be focused on the AFP's function in the soil.

Anti-drought

Introduction

Hyaluronic acid (HA) is a polysaccharide with a highly hydrating effect, and bacterial cellulose (BC) is a biological macromolecule with excellent biocompatibility and water retention. The crosslinked product of bacterial cellulose and hyaluronic acid, which exhibits excellent water retention performance, was selected as water-retentive material. The microflora proportional regulation gene circuit (please see Results for details), co-culture technique (please see Results for details), and hardware (please see Hardware for details) were developed to produce the crosslinked product continuously. In this part, we verified the performance of the cross-linked product in soil.

Construct the crosslinked product successfully

Bacterial cellulose and hyaluronic acid standard were mixed with 1,4-butanediol diglycidyl ether (BDDE) to synthesize a crosslinked product. As shown in Fig. 4, according to the different proportions of bacterial cellulose to hyaluronic acid, we prepared different crosslinked products (please see Experiment for details).

Fig. 4. Different proportion of bacterial cellulose to hyaluronic acid

After we harvested the crosslinked products, we characterized their performance using Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and field emission scanning electron microscope (FESEM) (please see Experiment for details). ATR-FTIR was employed to characterize the crosslinked products freeze-dried (Fig. 5). According to reference, the peak in 1028 cm^-1 represents the functional group of hyaluronic acid, while the peak in 2865 cm^-1 represents the functional group formed after the crosslinked reaction. As shown in Fig. 6a and b, it is very exciting that crosslinked products formed, which was consistent with that in the reference.

Fig. 5 Freeze-dried crosslinked products of bacterial cellulose and hyaluronic acid

Fig. 6 Characterization results of crosslinked product of bacterial cellulose and hyaluronic acid through ATR-FTIR. Cross-linked one kind of bacterial cellulose with 2% (BC_d/HA₂), 1% (BC_d/HA₁), and 0.5% (BC_d/HA_0.5) hyaluronic acid. Cross-linked the other kind of bacterial cellulose with 2% (BC_s/HA₂), 1% (BC_s/HA₁), and 0.5% (BC_s/HA_0.5) hyaluronic acid. ATR-FTIR: Fourier transform infrared spectroscopy.

After the success of cross-link, FESEM was used to characterize the structure of crosslinked products of bacterial cellulose and hyaluronic acid (please see Experiment for details). As shown in the FESEM image, we can clearly observe a tighter structure in the cross-linked product of bacterial cellulose and hyaluronic acid than that of the bacterial cellulose standard (Fig. 7a). The surface of the crosslinked product is smoother due to the crosslinking reaction (Fig. 7b). The image was consistent with that in the literature. We believe that such a structure will retain more water.

Fig. 7 Characterization results of crosslinked product of bacterial cellulose and hyaluronic acid through FESEM. a Bacterial cellulose standard. b Crosslinked product of bacterial cellulose and hyaluronic acid. FESEM: field emission scanning electron microscope.

As shown in Fig. 8, the crosslinked product of BC_d/HA₂ and BC_s/HA₂ showed a higher water-retention efficiency, indicating that bacterial cellulose with 2% hyaluronic acid is the best ratio.

Fig. 8 The water-retention efficiency of crosslinked product of bacterial cellulose and hyaluronic acid. Crosslinked one kind of bacterial cellulose with 2% (BC_d/HA₂), 1% (BC_d/HA₁), and 0.5% (BC_d/HA_0.5) hyaluronic acid. Cross-linked the other kind of bacterial cellulose with 2% (BC_s/HA₂), 1% (BC_s/HA₁), and 0.5% (BC_s/HA_0.5) hyaluronic acid.

The product of co-culture fermentation exhibited an excellent water retention performance in the soil

Since we have performed the crosslinking of bacterial cellulose and hyaluronic acid, we have learned more about bacterial cellulose / hyaluronic acid nanocomposites. Then, we carried out the co-culture fermentation of E. coli Nissle 1917 (EcNP for short) and E. coli BL21(DE3) which produce bacterial cellulose and hyaluronic acid respectively. Following that, we carried out an experiment to investigate the water-holding capacity of the crosslinked product from the co-culture fermentation. In this experiment, we used pure water as the negative control and the product of co-culture fermentation as the positive control, while adding the product of co-culture fermentation with pure water was set as the experiment group. (please see Experiment for more details). As shown in Fig. 9, we can clearly observe that the Δweight of the experiment group is higher than that of the negative control group after being placed in a dry environment (Humidity: 24%) at 37 ℃ 0.5 hour.

Fig. 9 The water-retention efficiency of co-culture product. Δweight _{experiment group} = the weight of soil with co-culture solution and pure water minus the weight of soil with co-culture solution, Δweight _{negative control} = the weight of soil with pure water minus the weight of soil.

Conclusion

The co-culture fermentation production has greater water-holding capacity than that of the soil without water retention material, which indicates that the product exhibits excellent performance as a water-retention material. Thus, this product can be used in the soil to protect the commercial crop from the damage of drought.