the CNC Carrier
To make full use of termites’ characteristic, trophallaxis, to eliminate a whole nest of termites, we have to ensure that the worker termites arrive their nest alive. Meanwhile, there should be enough time for them to complete the process of trophallaxis before the toxins function.
Thus, we design bacteria carriers self-assembled from the generated cellulose nanocrystals (CNCs). The Figure 1 abstractly introduces the process of self-assembly between bacteria and CNCs.
The functions of carriers
To ensure the worker termites’ safe arriving to the nest, the bacteria shouldn’t be digested until specific stimulus comes. Therefore, the bacteria will be embedded in nanomaterial which achieve the controlled release of bacteria by stimuli-responsiveness.
A carrier also acts as a protection, preventing the bacteria from being released into environment or simply being degraded without completing its mission.
Thus, the material of the carrier should exist for a period of time in termites’ guts but finally be digested by stimuli-responsiveness. It also should be thick enough to keep the bacteria from leaking and safe enough to be used in environment.
Using CNC as embedding nanomaterial
CNC is used as the embedding nanomaterial of our genetically modified organism because it meets all demands mentioned above.
CNC tends to form multiple hydrogen bonds with the surface of the cell so it achieves self-assembly easily. Termites are sure to eat the embedded bacteria because cellulose is the main composition of wood, which is the staple food of termites.
Meanwhile, the multiple attractive features of CNCs, such as their inherent renewability and sustainability, high biodegradability and biocompatibility, high strength, large specific surface area, and nanoscale dimension, have led to effective application already,which provides a proper condition to get bacteria embedded.
1 1 Zhou, J. et al. Synthesis of multifunctional cellulose nanocrystals for lectin recognition and bacterial imaging. Biomacromolecules 16, 1426-1432, doi:10.1021/acs.biomac.5b00227 (2015).
Theory and Method
Cellulose has specific structure feature, based on which we successfully achieve self-assembly between CNCs and bacteria in aqueous solution driven by multivalent interactions especially the multiple hydrogen bonds. This page will discuss the main theories and methods of CNC carriers.
Structure of cellulose
Cellulose is a linear chain of (1-4)-β-D-glucopyranana. Besides the β-(1-4)-glucosidic bond, the intrachain hydrogen bonding between hydroxyl groups and oxygens of the adjoining ring molecules also strengthen the linkage and stabilize the linear structure of the cellulose chain(Figure 1a).
In the parallel stacking of multiple cellulose chains, the Van der Waals and intermolecular hydrogen bonds play a significant role, which promote the forming of elementary fibrils that further aggregate into larger microfibrils (5-50 nm in diameter and several microns in length) (Figure 1b) .
The multivalent interactions stabilize the cellulose fibrils. Because of the existence of side chains, within these cellulose fibrils there are two kinds of regions where one kind is arranged in a highly ordered (crystalline) structure, and the other kind is disordered (amorphous-like). After acid hydrolysis dissolved the amorphous-like regions, we will get cellulose nanocrystals (CNCs) from the crystalline regions (Figure 1c) .
Figure 1 a) Schematics of (a) single cellulose chain repeat unit, showing the connection sites of the 1 - 4 linkage and intrachain hydrogen bonding (dotted line), (b) idealized cellulose microfibril showing one of the suggested configurations of the crystalline and amorphous regions, and (c) cellulose nanocrystals after acid hydrolysis dissolved the disordered regions. Copyright 2011, Royal Society of Chemistry.
Multivalent interactions between CNCs and bacteria
There are abundant binding sites on the surface of bacteria for CNCs. The lipopolysaccharides, forming the outer cell membrane of gram-negative bacteria, have plentiful hydroxyl groups to bind with the same functional groups of CNCs by multiple hydrogen bond interactions, while the Van der Waals participate a significant part in polymers as well. As for gram-positive bacteria, the peptidoglycans play an analogous role in the interactions with CNCs. Based on the properties of the surface of bacteria, we achieve self-assembly between the bacteria and the CNCs driven by multivalent interactions especially the multiple hydrogen bonds.
Moon, R. J., Martini, A., Nairn, J., Simonsen, J. & Youngblood, J. Cellulose nanomaterials review: structure, properties and nanocomposites. Chem Soc Rev 40, 3941-3994, doi:10.1039/c0cs00108b (2011).
In this page, we will show you the complete experiment section in details. The Figure 1 has clearly introduced the profile of our experimental.
CNCs were prepared from α-cellulose (25 μm, Aladdin, China). H2SO4 (98 %) was purchased from Zheshi, China, the materials of phosphate buffered saline (PBS) were purchased from Hushi, China. Dialysis membranes (3500 MWCO) were from USA. 200-mesh Cu grids were purchased from Electron Microscopy Sciences. Cell cultures and 6-well plates were purchased from Corning. Escherichia coli strains BL21(DE3) and DH5α were purchased from Vazyme. Streptomyces avermitilis strain ATCC31267 were purchased from China General Microbiological Culture Collection Center, CGMCC
Ultrasonic treatments were done by using a JY92-2D Vibracell ultrasonic processor (Scientz, China). Freeze-drying was dealt in an Alpha 2-4 Lyophilizer (Zhiyao, China). Centrifugation was carried out in an Avanti J-25 Centrifuge and an Allegra 64R Centrifuge (Beckman Coulter). UV absorbance was analyzed using a TU-1800 UV absorbance spectrophotometer (Puxitongyong, China). The bacteria were incubated in a THZ-C-1 incubator shaker (Peiying, China). Sterilization was carried out using a LDZX-75KB sterilizer (Shenan, China). Scanning electron microscopy (SEM) images were obtained using a S-3000N scanning electron microscope (Hitachi). Transmission electron microscopy (TEM) images were obtained using HT7700 (Hitachi) and JEM-1230 TEM microscopes (JEOL) operating at an accelerating voltage of 100 kV. The analysis of flow cytometry (FCM) was done by using a ACEA NovoCyte flow cytometry (ACEA biosciences, China).
Acid hydrolysis of cellulose
Cellulose was hydrolyzed at 40 °C with 8.75 mL of 50 wt % sulfuric acid/g of cellulose. To find the most proper time of acidolysis, We carried on a gradient experiment, setting up four groups of experiments whose hydrolyzed time are 1h, 2h, 3h and 4h, respectively. The hydrolysis was quenched by diluting 10-fold with cold DI water. The crystals were collected and washed once by centrifugation for 10 min at 9000 rpm and final solution was collected by centrifugation for 10 min at 5000 rpm. The solution was dialyzed in dialysis membranes against ultrapure water until the pH was neutral. 
Freeze-drying to get final product
Freeze-drying to get the solid CNC and solve it at 0.25 mg/mL. Crystal aggregates were disrupted by sonicating the suspension for 24 min under ice-bath cooling with a Vibracell ultrasonic processor.
Thermal Gravimetric Analyzer (TGA)
Samples were freeze-dried overnight and then subjected to heating scans from 30 to 800 °C, with a rate of 10 °C/min under a nitrogen atmosphere.
Bacteria Interaction Assays
E.coli and Streptomycete were separately cultured until an OD600 of 0.5 was attained. The grown bacteria were centrifuged at 12000 rpm for 2 min, and the precipitation was washed in PBS buffer for three times. To E.coli(OD600 = 0.5 A, 1 mL) in PBS, CNC(0.25 mg/mL, 1 mL) was added in a 6-well plate and they were incubated in an incubator shaker (250 rpm) at 37 °C for 2 h.
SEM and TEM observation
The CNC solution and E.coli aliquots (1 mL) incubated with CNC were added to Cu grids, respectively, and freeze-dried overnight. The samples were observed using TEM. Besides, we used SEM to study whether bacteria have an influence on the features of cellulose. We used E. coli aliquots (0.2, 0.4, 0.6, 0.8 mL, respectively) incubated with CNC. Then we put the samples on the sample stages and dried them overnight before we observed.
Dynamic Light Scattering (DLS)
DLS was performed at room temperature. We used suspensions of E.coli or E. coli aliquots (1 mL) incubated with CNC in PBS buffer solution (pH 7.0, 0.1 M). By means of analyzing different particle sizes, the multiple hydrogen bond interactions between the CNCs and E.coil (see below) can be proved indirectly.
Flow cytometry (FCM)
We used suspensions of E.coli or E. coli aliquots (1 mL) incubated with CNC in PBS buffer solution (pH 7.0, 0.1 M). And E.coli we used expressed fluorescent protein mcherry. The largest excitation wavelength of mCherry is 587nm and the largest emission wavelength of mCherry is 610nm. Channel PerCP-A was used to do the observation. Approximately 1,500,000 cells were calculated in each group with a medium speed.
1 Wang, Q., Zhu, J. Y. & Considine, J. M. Strong and optically transparent films prepared using cellulosic solid residue recovered from cellulose nanocrystals production waste stream. ACS applied materials & interfaces 5, 2527-2534, doi:10.1021/am302967m (2013).
2 Roman, M. & Gray, D. G. Parabolic focal conics in self-assembled solid films of cellulose nanocrystals. Langmuir 21, 5555-5561, doi:10.1021/la046797f (2005).
3 Zhou, J. et al. Synthesis of multifunctional cellulose nanocrystals for lectin recognition and bacterial imaging. Biomacromolecules 16, 1426-1432, doi:10.1021/acs.biomac.5b00227 (2015).