Team
1. Attribution
Collaboration
Our modeling can be divided into four parts. Firstly, in order to enable our project to be applied in life, we design a device placed in soil, which can attract and kill nematodes by modified engineering bacteria inside. Secondly, assuming there is a farmland, we take advantage of nematodes’ movement analogue simulation to find the best position where the device should be placed and the best concentration of attracting substance. Thirdly, according to this concentration, we obtain the best size of the device by calculating. At last, we establish a database to enlarge applied range of our method to kill other pests. We would appreciate that new synthetic biological and environment-friendly methods can be shared and improved with the science researchers all over the world.
Acknowledgement
Thanks:
Prof. Zhu Xudong, Prof. Yang Dong and Prof. Xiang Benqiong
Offered us much precious advice on the project.
Hao Xiaoran, Chi Xiaodong, Huo Liang, Ai Ying and Wang Xuan
Offered us advice on our experiments and helped us solve problems.
The College of Chemistry, Beijing Normal University
Helped us do GC-MS.
Prof. Yang Chonglin of Chinese Academy of Sciences
Offered us C.elegens and taught us how to cultivate them.
Prof. Li Hongmei of Department of Plant Pathology，Nanjing Agricultural University
Helped us to do Species identification of nematodes.
BIT-China & ZJU-China & FAFU-CHINA
Offered us some helpful advice and we also had a collaboration together.
Agricultural Service Center of Ceyu, Hebei Province
Helped us learn more information about the damage caused by local nematodes and villager’s attitude towards agricultural insecticide.
Shang Zhong National Middle School of Guizhou Province
Supported us in our voluntary education.
National Chiao Tung University &Peking University
Provided us opportunities to communicate with other teams.
Feng Xiaofei and her family in Hebei Province
Helped us get soybeans samples and did a survey in the locality.
Prof. Wang Yingdian
Discussed the bright future of our project with us.
Sponsors
Beijing Normal University (BNU), is a public research university located in Beijing with strong emphasis on basic disciplines of humanities and sciences. It is one of the oldest and most prestigious universities in China. Our school supported us to our iGEM Journey and provided us adequate resources to complete the competition. Students of different colleges in our school took part in our team, and all these colleges supported us very much.
Notebook
We designed parts rMpL and bace16 by adding pBAD promoter (BBa_K206000) and RBS(BBa_B0034) to the upstream region of rMpL gene and bace16 gene, and we committed the GENEWIZ company to synthesize the rMpL and bace16 gene segment.
We did the simple experimental operation such as the enlarge culture of bacteria, culture preservation and transformation again to be further familiar with the laboratory apparatus and the solexa. We transformed the plasmid rfp-pSB1C3(BBa_J04450) in DH5α and proceeded amplification to obtain the standard plasmid backbone.
We prepared some common used reagent such as TAE, antibiotics and LB cultural medium.
We designed the sequence of Limonene synthase (LS) that would be transformed to E.coli to express. Our designed sequences was based on the two cDNA sequences of LS expression sequence--Citrus unshiu CitMTSE1 for d-limonene synthase and Mentha spicata 4S-limonene synthase, together with prefix and suffix codes and restriction enzyme cutting site were sent to the company for optimum synthesis.
We obtained the plasmid of gene rMpL with PUC57 backbone in the form of dry power. We dissolved this dry power and inoculated it on LB solid cultural medium through which we obtained the monoclonal colonies. We inoculated 10 mL centrifuge tubes with the monoclonal colony to produce bacterium solution for preservation and plasmid extraction, and we named the obtained plasmid pUC57-rMpL.
We digested the plasmid PSB1C3-RFP sent by iGEM and the pUC57-rMpL with restriction enzyme separately and obtained the PSB1C3 backbone and rMpL gene segment which were connected together later on to assemble the plasmid PSB1C3-rMpL.
We prepared the L-limonene and D-limonene and the synthases expression E.coli (BL21,pGEX-4T-1-sls-gpps) without standardization of them both, which were cryopreserved with glycerol.
We prepared the BW25113 competent cells in terms of the component efficiency kit, and we detected their efficiency of transformation by transforming rfp-PSB1C3 plasmids of different concentration into the cells.
We transformed pSB1C3-rMpL into BW25113 competent cells to cultivate on the LB plate and inoculated 10 mL centrifuge tubes with the obtained monoclonal colony to produce bacterium solution for preservation and plasmid extraction.
We did restriction enzyme digestion to check the extracted plasmid.
We revived the limonene synthases expression E.coli strain preserved in glycerol.
We assembled bace16-pSB1C3 and transformed it into E.coli DH5α. After the result of digestion exam was correct we extracted the plasmid and did sequencing, and we transformed the plasmid with correct sequence into E.coli BW25113 to establish expression strain.
We activated the preserved culture and enlarge cultivated it in 250 mL conical flask, inducting expression with different concentration gradient of L-arabinose, we collected the precipitation after centrifuging the bacterium solution. We resuspended the precipitation with buffer A and after ultrasonication we obtained homogenate, then we did high speed centrifugation to get the supernatant. However, we regrettably observed that the effect of expression is not ideal from the SDS-PAGE gel electrophoresis of both the homogenate and the supernatant.
We fetched the N2 wild type caenorhabditis elegans favored by professor Yang Huilin of Institute of Genetics and Development Biology, Chinese Academy of Science, and studied the methods of cultivating nematodes as well as synchronization. Additionally, we learned the life history, growth properities and feeding method of nematodes.
We prepared he nematodes for enlarge cultivation and induced expression of the LS expression E.coli, and we synchronized a batch of nematodes for verification.
We digested rfp-pSB1C3 with two isocaudamas Spe I and Xba I and prepared empty vector plasmid as negative control of whether the desired gene expressed.
We prepared NGM cultural medium and cultivated OP50 E.coli in LB liquid medium.
We cultivated nematodes.
We cultivated nematodes and proceeded synchronization, and we did replication experiment of baiting nematodes with linalool using pure chemical compound (linalool). We optimized the protocol of replication experiment on and on, and the growth condition of nematodes is favorable. However, according to our statistical result we were not able to conclude that linalool can attract nematodes. And we processed nematodes’ chemotaxis replication experiment of limonene as well.
We also did expression replication experiments by dong SDS-PAGE under the protocol mentioned above (reserve the homogenate as protein sample) which came out an obvious band (89kd).
We ensured the empty vector plasmid was correct with three groups of restriction digestion.
We transformed bace16- pSB1C3 assembled by GENEWIZ and ourselves into the expression strain BW25113 to express bace16 as an attempt. We added 500 uL 1M L-Arabinose into 100mL bacterium solution to induce expression at 26C for 5h. We obtained proteins by precipitation with ammonium sulfate and concentration through dialysis which was dissolved with buffer A and then went through ultrasonication to get homogenate, and then high speed centrifugation for the supernatant. We did SDS-PAGE separately for the concentrated proteins, homogenate and supernatant. However, as the protein loading buffer was not commercial, the outcome effect was not satisfied.
| Group number | Nematodes amount(\(\mu\)L) | Sample amount(\(\mu\)L) | bace16 | Heated bace16 | pSB1C3 | M9 |
| 1 | 50 | 20 | Died immediately，and the cells lysed | Died immediately，and the cells lysed | Died immediately，and the cells lysed | Alive, active |
| 2 | 750 | 30 | Died totally while the morphology was completed. | -- | Died totally while the morphology was completed. | Partly died |
| 3 | 750 | 10 | Partly died | A few died, inactive | A few died, inactive | In good condition, active |
We repeated the induction experiment with different concentration gradient of arabinose as before, while we just collected the precipitation without extracting proteins this time.
We tried another protocol of replication experiment as increasing the amount of sample whereas the effect was still not ideal indicated by the statistical result.
We obtained gene l-limonene and d-limonene connected with pUC57 backbone in the form of dry power. We dissolved these dry power and transformed them into E.coli DH5α, coating on LB plate. We inoculated the obtained monoclonal colony into 10 mL centrifuge tubes to produce bacterium solution for preservation and plasmid extraction. The correct plasmids examined with restricton enzyme digestion was preserved in -20C, named l-limonene-PUC57 and d-limonene-PUC57.
We digested the plasmid pSB1C3-RFP from iGEM as well as l-limonene-PUC57 and d-limonene-PUC57 separately with restriction enzymes to get pSB1C3 backbone and the two gene segments, among which the brightness of pSB1C3 backbone band was quite weak. Then we recycled the backbone and gene segments following the agarose gel DNA extraction kit and linked them to produce plasmids l-limonene-PSB1C3 and d-limonene-PSB1C3.
We continued to watch the movement of nematodes under toxic test, where the nematodes of M9 group was generally active, partly died of bace16 and heated bace16 groups, and acted normally in pSB1C3 group.
Another SDS-PAGE of extracellular proteins extract was run while the objective band was still not observed. So we extracted the bace16-pSB1C3 from the expression strain BW25113 for restriction enzyme digestion and no objective band was extracted still. Meanwhile, it appeared to be some problems of other groups’ digestion too, against which we speculated that the cause may be the pollution of reagent, star activity or something wrong with the expression strain. We transformed into E.coli DH5α the bace16-pSB1C3 to find that the result of plasmid extraction and enzyme digestion was correct, thus we confirmed the problem was from the expression strain.
We re-cultivated the collected bacteria and mixed up the recombined bacteria with the control ones to a certain scale (1:4) to coat on NGM medium, preparing for nematodes cultivation of toxic test.
We transformed the obtained plasmids l-limonene-PSB1C3、d-limonene-PSB1C3 into E.coli BW25113 and cultivated 10mL centrifuge tube with them to produce bacterium solution for preservation and plasmid extraction. We examined the extracted plasmid with enzyme digestion, no objective band observed as a result.
We obtained gene l-limonene and d-limonene connected with pUC57 backbone in the form of dry power. We dissolved these dry power and transformed them into E.coli DH5α, coating on LB plate. We inoculated the obtained monoclonal colony into 10 mL centrifuge tubes to produce bacterium solution for plasmid extraction, the enzyme digestion result of which was incorrect that a band around 500~750 bp appeared whereas the backbone band lost. The problem was confirmed to be from the expression strain as mentioned above, so we adopted a new strain of BW25113 and continued our experiment.
We enlarge cultivated the standard LS expression bacteria (E.coli-BW2513-psb1c3) and expressed the objective produce (in contrast with empty vector). We did SDS-PAGE under the protocol mentioned above (reserve the homogenate as protein sample), no significant difference observed between the bands.
In addition, the differences between bands of BW25113 and BL21 was obvious when run SDS-PAGE together whereas the objective band of BW25113 cannot be ensured.
We expressed bace16 protein twice and prolonged the expression period to overnight. Also, we changed the concentration of L-Ara and increased the concentration of running gel to 15% when running SDS-PAGE. Still, we failed to observe objective band at all the supernatant of bacterium, homogenate and supernatant of ultrasonic broken cells.
We induced expression of rMpL continuously with arabinose in concentration gradient to acquire a stable expression condition. We extracted proteins to do SDS-PAGE, again finding no sign of rMpL expression.
Sequencing gene l-limonene and d-limonene synthesized by the company to get an unsatisfactory result, we speculated that these plasmids were extracted from the wrong BW25113. So we re-transformed the plasmids from the company into E.coli DH5αand BL21 separately and did all the cultivation, plasmid extraction, enzyme digestion de novo to make sure the examine result correctly. And we sent the plasmids extracted this time for sequencing.
We re-prepared the homogenate of the revived E.coli-BL21(pGEX-4T-1-sls-gpps) for SDS-PAGE and enlarge cultivated the standard bacteria (BL21-pxb1c3) transformed the second time to express the objective product.
We transformed bace16-pSB1C3 into the newly obtained BW25113 competent cells to express for another time, and examined the result with SDS-PAGE.
We went on the expression experiment of rMpL as above except that we added parallel controlled groups whereas canceled the control ones expressed under 37C and prolonged the expression period. However, we still failed to observe obvious expression of rMpL, against which we suspected there occurred something wrong with the expression bacteria and planed to change bacteria strain to express de novo.
We prepared NGM plate and nematodes liquid medium for toxic test of rMpL.
5. Protocol
5.1 Cloning
PCR
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Reaction System:
H2O 38 \(\mu\)L
10x Taq buffer 5 \(\mu\)L
2.5mM dNTP 4 \(\mu\)L
Primer 1 1 \(\mu\)L
Primer 2 1 \(\mu\)L
Template 0.5 \(\mu\)L
Taq 0.5 \(\mu\)L
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Process:
\(\begin{equation}\left. \begin{array}{lcl} {94°C\ 10min} \\ {94°C\ 30s} \\{58°C\ 30s} \end{array} \right\} Cycle\ 30\end {equation}\)
94°C 10min
94°C 30s
58°C 30s
72°C 2min30s
72°C 10min
10°C ---
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Material:
Agarose gel: 1% agarose dissolved in 1 x TAE + gelstain
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Protocol:
We used gelstain to stain the DNA and imaged it in a Transilluminator.
We used the gel extraction kit to get the objective fragment.
Electrophoresis---Gel Purification
Digestion
| 50\(\mu\)L reaction system | |||||
| Reagent | 10x H buffer | EcoR I | Pat I | Plasmid | H2O |
| Dosage | 5 \(\mu\)L | 1.5 \(\mu\)L | 1.5 \(\mu\)L | 15 \(\mu\)L | 27 \(\mu\)l |
| 10\(\mu\)L reaction system | |||||
| Reagent | 10x H buffer | EcoR I | Pat I | Plasmid | H2O |
| Dosage | 1 \(\mu\)L | 0.3 \(\mu\)L | 0.3 \(\mu\)L | 3 \(\mu\)L | 5.4 \(\mu\)L |
Ligation
| Ligation reaction system | ||||
| Reagent | Cph8 | PSB1C3 | T4 buffer | T4 ligase |
| Dosage | 14 \(\mu\)L | 3 \(\mu\)L | 2 \(\mu\)L | 1 \(\mu\)L |
5.2 Transformation
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Material:
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Protocol:
preparation of the competent cells
20\(\mu\)L ligation product + 50\(\mu\)L cells
Heatshock of E.coli BW25113(42°C,90s)
Put on ice(2min)
Add 800\(\mu\)L LB media and incubate for 1.5h(37°C, 150rpm)
Centrifuge at 4000rpm for 1min and remove 900\(\mu\)L supernatant
Resuspend the pellets using the left supernatant
Spread plates(with ampicillin)
Incubate for 12~16h(37°C)
| LB liquid medium | |||
| Reagent | Tryptone | Yeast extract powder | NaCl |
| Dosage | 10 g/L | 5 g/L | 10 \(\mu\)L |
5.3 Detetion
SDS-PAGE
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Materials:
Gel Tris-HCl Acr/Bis 30% SDS 10% ddH2O TEMED AP 10% Stacking Gel(4%) pH=6.8 500\(\mu\)L 500 \(\mu\)L 25 \(\mu\)L 1350 \(\mu\)L 2.5 \(\mu\)L 12.5 \(\mu\)L Running Gel(12%) pH=8.8 1250 \(\mu\)L 2000 \(\mu\)L 50 \(\mu\)L 1675 \(\mu\)L 2.5 \(\mu\)L 25 \(\mu\)L Running Gel(18%) pH=8.8 1250 \(\mu\)L 3000 \(\mu\)L 50 \(\mu\)L 675 \(\mu\)L 2.5 \(\mu\)L 25 \(\mu\)L Running Buffer Reagent Tris-HCl Glycine (w/v) SDS Dosage 25 mmol/L 0.192 mol/L 0.1% -
Protocol:
The SDS polyacrylamide gels are prepared in the so-called PerfectBlue™ Twin Double Gel System.
After ensuring that the equipment is waterproof, the 12 % (or 18%) running gel is mixed and filled into the chamber. Pipetting about 1 ml of H2O on top of the running gel to seal the gel.
After polymerization, the remaining H2O is removed and the 12 % stacking gel is filled on top. Insert a comb to create sample pockets.
After the stacking gel also polymerized, 1 x running buffer is used to run the Double Gel System via the SDS gel.
After loading the generated pockets with the samples, the stacking gel is run at 100 V and then running gel at 120 V.
Detection of Baiting Nematodes
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Materials:
Pure chemical compound: linalool and limonene.
Solvent: DMSO
Dissolve the compounds with DMSO to set a series of concentration gradient of attractant.
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Protocol:
Synchronize the nematodes.
Divide the NGM solid medium (d = 6 cm) equally into two parts (drawing on the surface of the culture dishes).
Put 50 \(\mu\)L compound of different concentration and DMSO as contrast respectively on the two parts. Make 3 repeats of each concentration.
Flush nematode from the plate with M9 and inoculate 20 \(\mu\)L (about 30 nematodes) into the center of the medium.
Incubate at 20°C for 1hr, and then place them at 4°C refrigerator for 1hr.
Count the nematodes of each part under the stereoscope.
Compare the results of different compound concentration and make a conclusion.
Detection of Killing Nematodes
Inoculate 5mL LB liquid mediums added Chl with experimental group E.coli BW25113 (transformed with rMpl or bace16 genes) and blank control group separately. Incubate at 37°C, 190 r.m.p for 3 hr.
Mix up 800\(\mu\)L control group and 200ul experimental liquid medium Inoculate 1mL mixture mentioned above to NGM plate. Incubate upside down at 37°C overnight.
Flush nematodes from the plate with M9, centrifugate (1500 r.p.m, 3min), abandon the supernatant and resuspend the precipitation with 3mL M9.
Add 500\(\mu\)L resuspended solution to the NGM plate inoculated with mixed E.coli as mentioned above as experimental group and NGM plate with blank E.coli as control group. 37°C incubate overnight.
Observe and compare the activation and size of nematodes of each group and draw a conclusion.
5.4 Nematode
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Materials:
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NGM medium:
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OP50 E.coli deficit type (food of nematodes):
| Minimum Medium | ||||
| Reagent | NaCl | Bacto peptone（BD 4059002) | Agar | ddH2O |
| Dosage | 3 g | 2.5 g/L | 17 \(\mu\)L | 975 mL |
Prepare 1mol/L CaCl2, 1mol/L MgSO4 and phosphoric acid buffer meanwhile.
| Phosphoric Acid Buffer | |||
| Reagent | KH2PO4 | KHPO4 | ddH2O |
| Dosage | 108.3 g | 35.6 g/L | 1 L |
Autoclave the above mentioned reagents with 120°C for 30 min and then water bath them to 65°C.
Dissolve the cholesterol with ethanol to 5 mg/mL.
Add 1ml cholesterol, CaCl2, MgSO4 and 25 mL phosphoric acid buffer into the minimum medium in order (all at 65°C, shake up).
Make plate with the NGM medium, storing at 4°C.
Streak inoculate OP50 on LB solid medium, Incubate at 37°C for 6 hr or overnight. Inoculate LB liquid medium with OP50 single colonies, and incubate (37°C, 220 r.p.m) overnight.
Cultivate
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The incubation condition of caenorhabditis elegans N2 wild type:
Generally incubated at 20°C. Grow slowly at 16°C and grow fast at 25°C while egg laying amount declines.
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Protocol:
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Materials:
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Protocols:
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modified Baermann funnel method:
Inoculate NGM medium with 150~200 \(\mu\)L OP50, incubate at 37°C for 12hr. Cut down a square of NGM(about 1cm x 1cm) with nematodes.
Put the square on the NGM medium with OP50 and let the surface with nematodes adown to contact the medium.
Seal and incubate upside down at 20°C.
Watch the growth condition of nematodes under the stereoscope everyday and re-inoculate every 4 to 5 days to avoid the nematodes growing too densely.
For inoculating abundant nematodes rapidly, or changing plate to provide more food and better condition for them, we can flush them with M9 and centrifuge them with 1500 r.m.p for 1 min, and incubate the precipitate on a new plate.
Synchronization
| M9 NS | |||||
| Reagent | Na2HPO4 | KH2PO4 | NaCl | 1M MgSO4 | ddH2O |
| Dosage | 6 g | 3 g | 5 g | 1 mL | 1 L |
| Bleach Buffer | |||
| Reagent | NaOCl | 5M NaOH | ddH2O |
| Dosage | 50 \(\mu\)L | 100 \(\mu\)L | 850 \(\mu\)L |
We have tried several methods and two of them succeed as mentioned below, we recommend the first one by comparison.
1. Flush and incubate
Flush the nematodes with M9 NS
Centrifugation (3000 r.m.p, 1min)
Abandon the supernatant, add 1 mL bleach buffer to the precipitation
Centrifugation (3000 r.m.p, 1min), abandon the supernatant
Transfer the precipitation (nematode eggs) to a NGM plate with OP50, then the eggs will incubate synchronously.
2. Directly pick
Transfer 30 nematodes that are under egg laying period to a new NGM plate with OP50. Generally, the egg laying nematodes can lay about 8 eggs per hour. Remove all nematodes after 2hr.
Incubate the plate at 20°C for 3d and therefor can get about 300 to 400 nematodes.
Note: all of the procedure outlined above must be conducted under sterile condition.
Isolate nematodes from soil
1. Place a glass funnel (d = 10~15 cm) on the funnel stand linked with about 10cm rubber tube which is furnished with a flatjaw pinchcock.
2. Cut the soil sample into matchstick shape. Package 10 g(wet weight) with gauze and put it into the funnel, and then add water to immersion the sample.
3. After 4~24 hr, nematodes leave the plant tissue and swim in the water and ultimately precipitate at the bottom due to their gravity and hydrotaxis.
4. Open the flatjaw pinchcock, adopt about 5~15 mL liquid sample from the bottom which obtains most active nematodes of the soil sample. Check under the stereoscope and count nematodes, or centrifugate(1500 r) first and check the precipitation if the nematodes are too few.