iGEM REPORT: Termite Terminator: Genetically modified toxin protein expression systems with cellulose nano-crystal delivery carriers for trapping termites based on trophallaxis

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Termite Terminator: Genetically modified toxin protein expression systems with cellulose nano-crystal delivery carriers for trapping termites based on trophallaxis

Ziyang Gao (1,3), Yang Su (1,2), Chang Liu (1,4), Qianyun Zhang (1,2), Jianzhong Hu (1,2), Xintian Xu (1,2), Jiahao Gao (1,2), Hao Feng (1,10), Xukang Shen (1,5), Mengmeng Xu (1,6), Yue Zhong (1,7), Yuqing Shen (1,8), Tianning Zhang (1,9), Kunxun Qian (1,2), Yanruide Li (1,2), Ming Chen (2)*

(1) Zhejiang University Team (ZJU-China) for the International Genetically Engineered Machine Competition (iGEM)

(2) College of Life Sciences, Zhejiang University

(3) School of Basic Medical Science, Zhejiang University

(4) College of Chemical and Biological Engineering, Zhejiang University

(5) College of Agriculture and Biotechnology, Zhejiang University

(6) College of Optical Science and Engineering, Zhejiang University

(7) College of Electrical Engineering, Zhejiang University

(8) College of Environmental and Resource Sciences, Zhejiang University

(9) Department of Physics, Zhejiang University

(10) Department of Chemistry, Zhejiang University

* mchen@zju.edu.cn

Author contributions

Conceptualization: Z. Gao, C. Liu, J. Gao

Methodology: Y. Su, Q. Zhang, J. Hu, H. Feng

Investigation: Z. Gao, Q. Zhang, J. Hu, X. Xu, Y. Shen, J. Gao, H. Feng, X. Shen, Y. Su, K. Qian, Y. Li, M. Xu, Y. Zhong, T. Zhang

Writing—Original Draft: Z. Gao

Review & Editing: Z. Gao, X. Xu

Sponsor Funding Acquisition: C. Liu, X. Xu, J. Gao, Z. Gao

Supervision: M.Chen

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Abstract

In order to treat the termite issue using an environment-friendly method, we created a system named Termite Terminators, based on termites’ intrinsic habit—-Traphollaxis, which means that worker termites go out of the nest to seek food and feed the other roles of termites. First, We used synthetic biology principles to establish a toxin protein expression system in Escherichia Coli. The toxin proteins that we chose were originally discovered in Photorhabdus Luminescenes, and have been verified to be capable of killing other kinds of insects. Second, we designed a bacteria carrier system that self-assembled from the generated cellulose nano-crystals (CNCs).

By the function of bacteria carrier, we can make full use of termites’ trophollaxis to eliminate the whole nest. For the worker termites that search for food, once baited by our product, they will carry the product in the mouth instead of deglutition. Because of the low concentration of cellulase and lysozyme in termite’s mouth, the shell of bacteria carrier cannot be digested, so that the toxin proteins can stay unreleased in worker termites’ mouth. When worker termites go back to the nest, they will feed the product to soldiers, young, queen and king termites. When the product is swallowed in their guts, the cellulose carrier will be digested by the high concentration of celluase and lysozyme, and the toxin can be released in their gut, so that the whole nest can be killed by the accumulated effect of Termite Terminator.

Introduction

Termites are a group of insects that are classified as the infraorder Isopteta, and they are widely recognized as the 5^th pest around the world as they can seriously damage the wooden structure of buildings and house furnishings. The traditional ways of controlling termites are mostly in two aspects: chemical pesticides and baiting system. However, people began to realize that using chemical pesticides is not an environmental way and the pesticide residual can be harmful to human being’s health. As a result, we tried to utilize methods of synthetic biology to establish a more environmentally friendly way for termite control based on their intrinsic habit – traphollaxis. Trophollaxis is the transfer of food from worker termites to other kinds of members of termites through mouth-to-mouth.

With the purpose of killing termites, we chose several kinds of toxin proteins respectively bacillus thur-ingiensis(bt)-like Plu0840 and enterotoxin-like Plu1537. The host of these proteins is Photorhabdus Luminescens TT01, a kind of gram-negative bacteria, which is capable of producing and releasing a variety of inscrtifal and bactericidal toxins.[1] Plu1537 is a bt homologous toxin protein, exact function of which is still unclear, but Li’s research in 2009 indicated that Plu1537 had insecticidal activity.[2] Plu0840 is an enterotoxin Ast homologous protein. Plu0840 is confirmed to have oral toxicity against several kinds of insects.[3] We constructed the pSB1C3 carried toxin proteins plasmid respectively by the principle of rfc10 and submitted the Biobrick parts to iGEM Biobrick registry.

To ensure that worker termites can carry our product to the nest safely and other termites in the nest can be killed by toxin protein, we used nanomaterial technology to design the artificial cellulose shell of bacteria carrier. Cellulose is a linear chain of (1-4)-β-D-glucopyracana. Besides the β-(1-4)-glucosidic bond, the intra-chain hydrogen bonding between hydroxyl groups and oxygens of he adjoining ring molecules also strengthen the linkage and stabilize the linear structure of the cellulose chain. 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. 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. [4] Because the lipopolysaccharides, forming the outer cell membrane of gram-negative bacteria, have plentiful hydroxyl groups to bind with the same functional groups of CNCs, nano-CNC solution tends to form multiple hydrogen bonds with the surface of the cell. The system can achieve self-assembly driven by multivalent interactions, while Van Der Waals participate a significant part in polymers as well.

Methods and Materials

1. Toxin proteins manufacture

The aim of toxin proteins manufacture is to establish an expression system of the two chosen toxin proteins from Photorhabdus Luminescenes TT01 in E.coli Bl21.

1.1 Strains and Plasmids

• Primary host organism: Photorhabdus Luminescenes TT01
  • The Photorhabdus Luminescenes TT01 strain was kindly given by Prof. Y.Zhang of Shandong University.
• Expression host organism: coli BL21
  • The E.coli BL21 strain was kept in our iGEM laboratory in -80°C refrigerator.
• Engineered construction host organism: coli DH5α
  • The E.coli DH5α strain was kept in our iGEM laboratory in -80°C refrigerator.
• Plasmid backbone: pSB1C3
  • The backbone was given by iGEM foundation in parts distribution.

1.2 Media and culture conditions

Photorhabdus Luminescenes TT01 was grown at 37°C in Luria-Bertani(LB) medium(120rpm,30-36 hours). E.coli BL21 and E.coli DH5α were both grown at 37°C in Luria-Bertani(LB) medium(120rpm,12-16 hours) and chloromycetin (50μg/ml) was added when necessary for plasmid propagation.

1.3 DNA manipulation and sequencing

Chromosomal DNA of Photorhabdus Luminescenes TT01 were isolated by using Bioteke Bacterial DNA Extraction Kit. Plasmid DNA of E.coli BL21 and E.coli DH5α were extracted by Axygen miniPrep Kit. PCR was carried out using PrimeStar HS DNA Polymerase. DNA sequencing was performed by Tsingke Biotechnology Co.(Hangzhou) The plasmid DNA was constructed by scarless assembly method using MultiS One-step Cloning Kit of Vazyme Co.

1.4 Construction of toxin protein plasmids

We chose pBad/araC as the promoter and the toxin proteins as the genes of interest and mCherry as the reporter gene, with RBS adding in front of segments need to be expressed.(Fig 1a) The genome sequence of Photorhabdus Luminescenes TT01 was downloaded from Genbank-AY526326. mCherry and pBad segments were achieved in iGEM parts distribution. The toxin protein plasmid was constructed as follows: Using genomic DNA of Photorhabdus Luminescenes TT01 as template, Plu0840, Plu1537 and mCherry were amplified by PCR. pBad was amplified by PCR, RBS adding behind, with the template as BBa_I0500 part given by iGEM distribution. Then pBad-Plu0840-mCherry and pBad-Plu1537-mCherry were respectively constructed into pSB1C3 backbone by the One-Step Cloning. Then we got the biobrick-principled plasmids.(Fig 1b and 1c) The detailed sequence of these two plasmids can be found in the parts page of our submission(BBa_K1668010 and BBa_1668009).

1.5 Measurement

Reporter gene expression and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue (CBB) staining was carried out for measurement of toxin protein expression.

 

Fig. 1 Circuits designing of toxin proteins a) Toxin proteins circuits designed by ZJU-CHINA 2015 to produce Plu_0850 and Plu_1537 in coli BL21 b) Plasmid profile of submitted generator Biobrick: BBa_K1668009 which is capable of producing Plu_0840 in coli BL21 c)Plasmid profile of submitted generator Biobrick: BBa_K1668010 which is capable of producing Plu_1537 in coli BL21

2. Bacteria Carrier

The aim of bacteria carrier manufacture is to let the toxin produced E.coli be embedded in the cellulose nano-crystals, so that enveloped bacteria can function as the carrier in order to terminate the termites.

2.1 Materials preparation

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.

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.

2.2 CNC preparation methods

The CNC preparing methods were generated by Hao Feng of ZJU-China 2015 Team.(Fig 2)

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. [5]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.[6]

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.

Fig. 2 Flow chart of producing liquid CNC solution

 

2.3 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.

2.4 Bacteria Interaction Assays

E.coli was 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.[7]

2.5 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.

2.6 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.

Results

Toxin proteins expression in E.coli BL21

To confirm the successful construction of the toxin plasmids, we electrophoresed both the single and double digestion product of cloned mCherry (Biobrick-Coding Sequence), Plu_0840 (Biobrick-Device) and Plu_1537 (Biobrick-Device) shown as below. (Fig 3a-c)

Red colonies of Plu_0840 and Plu_1537 were picked respectively from plates with 24-hour cultured in 80mM/L arabinose and 50μg/ml chloromycetin LB solid medium, and the successful expression of reporter gene—-mCherry indicates that the Plu_0840 and Plu_1537 are successfully expressed. (Fig 3d) For further verification of the protein expression, we did SDS-PAGE and CBB Staining. We observed obvious expression of mCherry, Plu_0840 and Plu_1537. (Fig 3e)

 

Fig. 3 Toxin proteins expression in E.coli BL21 a)DNA electrophoretogram of the single and double digestion product of cloned mCherry (Biobrick-Coding Sequence) b) DNA electrophoretogram of the single and double digestion product of cloned Plu_0840(Biobrick-Device) c) DNA electrophoretogram of the single and double digestion product of cloned Plu_1537(Biobrick-Device) d) Bacteria in pipets were coli BL21 control, BL21 with Plu_1537 device, BL21 with Plu_0840 device respectively. The Expression of reporter gene mCharry indicates the expression of Plu_1537 and Plu_0840 are expressed in E.coli BL21. e) The proteome SDA-PAGE with CBB Staining of BL21 with Plu_1537 device, Plu_0840 device, mcherry and control respectively. The figure shows that the bacteria containing with plasmid of Plu_1537 device or Plu_0840 device co-expressed mCherry and toxin proteins together.

Bacteria Carrier of CNC

CNC Preparation

According to the quenching effect from 10-fold cold DI water, we got the raw product solutions. With abundant raw cellulose fibers existing, the solutions are a little turbid. After centrifugation, we prepared the CNC Suspension. We used red laser pointer to irradiate DI water and the CNC Suspension, respectively, and only the CNCs form the Tyndall effect, which proved the existence of CNC conveniently. (Fig 4b) After overnight freeze-drying process, the final product of CNC was obtained in the beakers as the powder form. (Fig 4c) TGA was carried out to observe the thermal characteristics of the CNC. (Fig 4d) Evaporation of water led to the first stage of gradual weight loss. The onset temperature which CNCs began to degrade was around 223°C. The most obvious weight loss occurred at 393°C while the literature value is 313°C, indicating the high thermal stability of CNCs we made.[7]

Fig. 4 results of CNC preparation a) Raw product solutions of CNC b) Red laser pointer shows the Tyndall effect of the CNC solution. Meanwhile, DI water as the negative control cannot from Tyndall effect. c) Product of solid pure CNC after overnight freeze drying d) TGA result demonstrates that the onset temperature which CNCs began to degrade was around 223°C. The most obvious weight loss occurred at 393°C while the literature value is 313°C, indicating the high thermal stability of CNC we made.

Observation of CNC Bacteria Carrier

The pure CNC is observed to crystallize in aqueous solutions and thus forms a square shape under TEM, which can be a standard to recognize whether bacteria are embedded in the CNC. (Fig 5e)

In the Fig 5a, 5b taken under TEM, it’s obvious that the fibers of CNC are attached to the surface of E.coli, which reveals that the CNCs have successfully wrapped E.coli. Meanwhile the profile of CNCs has been displayed in Fig 5c, its sphere is extremely smooth while that of CNCs with E.coli is relatively rough. The red arrow of Fig 5d clearly indicates the location of E.coli. (Fig 5a-d)

The Dynamic Light Scanning (DLS) reveals the embedding situation of E.coli with CNCs. E represents the pure E.coli. 4 h-CNC-E represent the microsphere of E.coli with CNC. (Fig 5f) The result of DLS indicates that 4 h-CNC-E occurs obviously self-assembly in general. The average particle sizes of each kind of compound are shown on the Table 1. Through simple subtraction, we can get the thickness of CNC on the surface of E.coli: Thickness = (1513.8 – 1317.1)/2 = 0.9835 nm.

Table 1 The average sizes of compounds

Fig. 5 TEM and SEM observation and DLS of CNC bacteria carrier a) and b) TEM images of CNC capsulated Bacteria Carrier. The fibers of CNC are attached to the surface of coli. Fig 5b shows the clustered form of E.coli with CNC capsulated. c) SEM image of CNC self-assembly. The sphere of pure CNC cluster is smooth. d) SEM image of CNC wrapped coli. The red arrow clearly shows the location of E.coli. e) TEM image of pure CNC crystallization. The pure CNC crystallizes in aqueous solutions and forms a square shape. f) DLS result of the embedding situation of coli with CNCs. E represents the pure E.coli. 4 h-CNC-E represent the microsphere of E.coli with CNC.

Discussion

During the time from February to October 2015, team members in ZJU-China 2015 devoted to find a better solution for termite issue based on the synthetic biology principles. We have successfully constructed the standardized Biobrick parts for the necessary toxin proteins expression system in E.coli. In addition, we created the bacteria carrier by the CNC shell capsulated out of the surface of E.coli. It’s the first time for synthetic biology and nanometer material technology be used in termite control, and we hope to see the further development of this method to have a better application in the real world. In order to analyze and forecast the effect of Termite Terminator in the reality, we made 3 models for termite trace simulation, mass transfer of bacteria carrier and degradation of the CNC shell, and the outcomes of modeling fit well in our wet-lab work (see more details for the models).

The function of the toxin proteins that we chose has been primarily tested in our 2015 iGEM work(see the detail of primary test result) and we hope that the mechanism of these toxin proteins can be unlocked in near future. However, because time is limited, we haven’t managed to explore the appropriate formula for our product and termite’s bait to have the best effect of terminating termites. We are looking forward to seeing the deeper research and the pest-control method based on combination of synthetic biology and material chemistry better developed.

Supporting Information

Supplemental Results

References

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[2] Li M, Wu G, Liu C, et al. Expression and activity of a probable toxin from Photorhabdus luminescens[J]. Molecular biology reports, 2009, 36(4): 785-790. doi: 10.1007/s11033-008-9246-z. pmid: 18409059

[3] Li M, Qiu L, Pang Y. Cloning and expression analysis of a predicted toxin gene from Photorhabdus sp. HB78[J]. Annals of microbiology, 2007, 57(3): 313-319. doi: 10.1007/BF03175066

[4] Moon R J, Martini A, Nairn J, et al. Cellulose nanomaterials review: structure, properties and nanocomposites[J]. Chemical Society Reviews, 2011, 40(7): 3941-3994. doi:10.1039/c0cs00108b (2011). pmid: 21566801

[5] 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[J]. ACS applied materials & interfaces, 2013, 5(7): 2527-2534. doi: 10.1021/am302967m. pmid: 23473973

[6] Roman M, Gray D G. Parabolic focal conics in self-assembled solid films of cellulose nanocrystals[J]. Langmuir, 2005, 21(12): 5555-5561. doi:10.1021/la046797f (2005). pmid: 15924489

[7] Zhou J, Butchosa N, Jayawardena H S N, et al. Synthesis of Multifunctional Cellulose Nanocrystals for Lectin Recognition and Bacterial Imaging[J]. Biomacromolecules, 2015, 16(4): 1426-1432. doi: 10.1021/acs.biomac.5b00227. pmid: 25738860

 

Acknowledgements

The author would like to thank all members of historical ZJU-China iGEM Team. We especially thank teacher Ming Ding for the kind support to every iGEMer of Zhejiang University for more than 6 years. We thank Prof. Jianchu Mo, Dr. Han Li and Dr. Shiyou Liang of Institute of Insects Science Zhejiang University for informing us with the basic knowledge of termites. We thank Dr. HanLi of Institute of Biochemistry Zhejiang University of teaching us the skills of molecular biology. We thank Dr. Yu of Department of Chemistry Zhejiang University for his instrument of CNC preparation. We thank all the people who have helped ZJU-China during the most meaningful days in iGEM competition.

 

Financial Disclosure

The funders had no role in study design, data, collection and analysis, decision to publish, or preparation to the manuscript.

 

Competing Interests

The authors have declared that no competing interests and financial exist.

 

Ethics Statement

N/A

 

Data Availability

Yes – all data are fully available without restriction