Difference between revisions of "Team:BNU-CHINA/Team"

Revision as of 04:11, 14 September 2015

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Team:BNU-CHINA - 2015.igem.org

1. Attribution

Project Design and Wet Lab Experiments

Under the supervision of Prof. Zhu Xudong, Prof. Yang Dong, and Prof. Xiang Benqiong etc, nearly all team members have taken part in the project designing process, especially those of wet lab group.Beichen Gao , Yuanyi Dai , Yufei Cao and Jia Li are the group leaders who mainly design the experimental patterns and guide the different parts of the experiments.

Modeling

Cheng Li is the leader of modeling group, who mainly enrolled in designing the device 2.0. Yuan Yin , Qiuyue Yuan and Xueting Zhao also do a great many works in the modeling group, such as information searching, paper writing and equipment appilication.

Wiki Construction

Jiajun Zhang , who has led the web design group to build the wiki, together with group members Zhiyao Chen and Chengfei Peng complete the main work in database construction and maintenance.

Graphic design

Lu Xu( group leader), Lee and Yalei Cao really do a fantastic work in graphic and web design. They all make great efforts in designing our team logo, website, poster, souvenir, T-shirt and so on.

Human practice

Meng Tang , the leader of human practice group, has organized a series of activities for Policy and Practice, like survey and publicize in some middle schools etc. Some group members as Yuanyi Dai,Jia Li , Jiajun Zhang , Beichen Gao , Zhiyao Chen , Jingyu Zhao , Xiaofei Feng also play a significant role in planning and organizing the activities . Other group members also take an active part in some other activities of human practice.

2. 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.

3. 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.

4. Notebook

Week 1

We found two kinds of toxic proteins that can kill nematodes specially—Bace16 and MpL—through reviewing literatures which confirmed the function of poison nematodes of these two proteins. We found the gene sequence of bace16(AAV3D0845) and the cDNA sequence of MpL(GeneBank accession number HQ449739) from the Genebank, and optimized the coded sequences in E.coli.

After we determined the theme of our project, we recuited more undergraduates from related colleges, including college of Mathematics, Physics and Computer Science. After a short meeting, four undergraduates joined our team. Congratulations!

Week 2

We tried some simple experimental operation such as the enlarge culture of bacteria, culture preservation and transformation to be familiar with the laboratory apparatus and the solexa.

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.

Modeling group members read some related materials and papers, including wikis of former teams especially their modeling parts as well as papers introducing nematodes.

Week 3

There occurs some problems of rMpL and bace16 gene synthesis. GENEWIZ gave us the relevant feedback in time and modified the two sequences to synthesize de novo.

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.

Modeling group members read some related materials and papers, including wikis of former teams especially their modeling parts as well as papers introducing nematodes.

Week 4

We extracted the plasmid and did restriction enzyme digestion to test and verify the plasmid backbone PSB1C3, and preserve the correct plasmid for subsequent experimental use.

Week 5

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.

Together with members majoring in physics, mathematics and biology, we drafted an abstract settlement concentrating on how to simulate the concentration of nematodes in chemotaxis. The goal of our modeling project is to give a best attractant of concentration so that we can determine the most economical plan.

Week 6

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.

Week 7

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 realized that our original assumption that we regard nematodes as static models cannot react to some essential properties of nematode moving. So we changed our minds into using dynamic methods to simulate the diffusion of nematodes in chemotaxis. We decided to apply Celluar Automata to solve the problem.

Week 8

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.

Since we can get the solution of best concentration of attractant and the diffusion of nematodes, we decided to go towards a further step. What is needed urgently in agriculture product is a low-cost and more importantly environment-friendly device to kill nematodes. Finally our plan was to design and build a debug device.

Week 9

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 built a device all by handwork! There was a big change in the process. At first, we wanted to design in CAD software and built it by 3D print. However, the cost of product by 3D print was too high. Finally, we chose traditional technology to produce devices in the future if it was verified that our device had high value of application. Of course, we also cannot afford the cost of custom-built sample. That’s why we had to make it by hand.

Week 10

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.

Another important module of our project is to build a database which researchers and even farmers can get information of bio-pesticide easily. We started to work on it.

Week 11

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.

We reviewed the work we had done together and made a specific plan for following days. First, we had built simulation model and we can use it to build economic model. Second, the device we had made actually was not applicable enough, which required further improvement. Third, we had built a basic frame of the database.

Week 12

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.

We put the database on the github.com and it can be edited and added new items by all users.

5.1 Cloning

PCR

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
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 ---

Electrophoresis---Gel Purification

Material:

Agarose gel: 1% agarose dissolved in 1 x TAE + gelstain
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.

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

Material:

LB liquid medium
Reagent	Tryptone	Yeast extract powder	NaCl
Dosage	10 g/L	5 g/L	10 $\mu$L

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)

5.3 Detetion

SDS-PAGE

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

Materials:

Pure chemical compound: linalool and limonene.

Solvent: DMSO

Dissolve the compounds with DMSO to set a series of concentration gradient of attractant.

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

Materials:
NGM medium:

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.

OP50 E.coli deficit type (food of nematodes):

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

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.

Protocol:

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

Materials:

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

Protocols:

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

modified Baermann funnel method:

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.

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

@@ Line 64: / Line 64: @@
          </nav>
      </header>
       <div class="content container">
          <sidebar class="col-md-3 bs-docs-sidebar">
@@ Line 105: / Line 106: @@
              </header>
              <section id="section-1" class="group">
+               <div class="content container">
+        <sidebar class="col-md-3 bs-docs-sidebar">
+            <ul id="sidebar" class="nav nav-stacked fixed">
+                <li><a href="#section-1">Attribution</a>
+                </li>
+                <li>
+                    <a href="#section-2">Collaboration</a>
+                </li>
+                <li>
+                    <a href="#section-3">Aknowledgement</a>
+                    <ul class="nav nav-stacked">
+                        <li><a href="#section-3-1">Thanks</a></li>
+                        <li><a href="#section-3-2">Sponsors</a></li>
+                    </ul>
+                </li>
+                <li>
+                    <a href="#section-4">Notebook</a>
+                </li>
+                <li>
+                    <a href="#section-5">Protocol</a>
+                    <ul class="nav nav-stacked">
+                        <li><a href="#section-5-1">Cloning</a></li>
+                        <li><a href="#section-5-2">Transformation</a></li>
+                        <li><a href="#section-5-3">Detection</a></li>
+                        <li><a href="#section-5-4">Nematode</a></li>
+                    </ul>
+                </li>
+            </ul>
+        </sidebar>
+        <article class="col-md-9">
+            <header class="page-header">
+                <h1>Team    <small>Beijing Normal University</small>
+            </h1>
+            </header>
+            <section id="section-1" class="group">
                  <h3>1.  Attribution</h3>
                  <div class="panel-group" id="accordion">
@@ Line 390: / Line 429: @@
                          <img src="https://static.igem.org/mediawiki/2015/2/25/BNU-bace16.png" style="float:left;">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.
                          <br/>
-                         <br/><img src="ihttps://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png" style="float:left;">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.
+                         <br/><img src="https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png" style="float:left;">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.
                          <br/>
                          <br/>
@@ Line 601: / Line 640: @@
              </section>
-             <section id="section-5" class="group">
+             <section id="section-5 " class="group ">
-                 <div id="section－5-1" class="subgroup">
+                 <div id="section－5-1 " class="subgroup ">
                      <br/>
                      <hr/>
@@ Line 611: / Line 650: @@
                          <li>
                              <h5>Reaction System:</h5>
-                             <p>H<sub>2</sub>O 38 \(\mu\)l</p>
+                             <p>H<sub>2</sub>O 38 \(\mu\)L</p>
-                             <p>10x Taq buffer 5 \(\mu\)l
+                             <p>10x Taq buffer 5 \(\mu\)L
                              </p>
-                             <p>2.5mM dNTP 4 \(\mu\)l</p>
+                             <p>2.5mM dNTP 4 \(\mu\)L</p>
-                             <p>Primer 1 1 \(\mu\)l</p>
+                             <p>Primer 1 1 \(\mu\)L</p>
                              <p>Primer 2 1 \(\mu\)L
                              </p>
@@ Line 626: / Line 665: @@
                              <h5>Process:</h5>
                              <p>\(\begin{equation}\left. \begin{array}{lcl} {94&#176;C\ 10min} \\ {94&#176;C\ 30s} \\{58&#176;C\ 30s} \end{array} \right\} Cycle\ 30\end {equation}\)</p>
-                             <p>94&#176C 10min
+                             <p>94&#176;C 10min
                              </p>
-                             <p>94&#176C 30s
+                             <p>94&#176;C 30s
                              </p>
-                             <p>58&#176C 30s
+                             <p>58&#176;C 30s
                              </p>
-                             <p>72&#176C 2min30s
+                             <p>72&#176;C 2min30s
                              </p>
-                             <p>72&#176C 10min
+                             <p>72&#176;C 10min
                              </p>
-                             <p>10&#176C ---
+                             <p>10&#176;C ---
                              </p>
                          </li>
@@ Line 656: / Line 695: @@
                          <h5 align='center'>Digestion</h5>
-                         <table class="table table-condensed">
+                         <table class="table table-condensed ">
                              <tbody>
                                  <tr>
-                                     <td>50\(\mu\)l reaction system</td>
+                                     <td>50\(\mu\)L reaction system</td>
                                      <td></td>
                                      <td></td>
@@ Line 677: / Line 716: @@
                                  <tr>
                                      <td>Dosage</td>
-                                     <td>5 \(\mu\)l</td>
+                                     <td>5 \(\mu\)L</td>
-                                     <td>1.5 \(\mu\)l</td>
+                                     <td>1.5 \(\mu\)L</td>
-                                     <td>1.5 \(\mu\)l</td>
+                                     <td>1.5 \(\mu\)L</td>
-                                     <td>15 \(\mu\)l</td>
+                                     <td>15 \(\mu\)L</td>
                                      <td>27 \(\mu\)l
                                      </td>
@@ Line 686: / Line 725: @@
                              </tbody>
                          </table>
-                         <table class="table table-condensed">
+                         <table class="table table-condensed ">
                              <tbody>
                                  <tr>
-                                     <td>10\(\mu\)l reaction system</td>
+                                     <td>10\(\mu\)L reaction system</td>
                                      <td></td>
                                      <td></td>
@@ Line 707: / Line 746: @@
                                  <tr>
                                      <td>Dosage</td>
-                                     <td>1 \(\mu\)l
+                                     <td>1 \(\mu\)L
-                                         <td>0.3 \(\mu\)l</td>
+                                         <td>0.3 \(\mu\)L</td>
-                                         <td>0.3 \(\mu\)l</td>
+                                         <td>0.3 \(\mu\)L</td>
-                                         <td>3 \(\mu\)l</td>
+                                         <td>3 \(\mu\)L</td>
-                                         <td>5.4 \(\mu\)l
+                                         <td>5.4 \(\mu\)L
                                          </td>
                                  </tr>
@@ Line 719: / Line 758: @@
                          <h5 align='center'>Ligation</h5>
-                         <table class="table table-condensed">
+                         <table class="table table-condensed ">
                              <tbody>
                                  <tr>
@@ Line 737: / Line 776: @@
                                  <tr>
                                      <td>Dosage</td>
-                                     <td>14 \(\mu\)l
+                                     <td>14 \(\mu\)L
-                                         <td>3 \(\mu\)l</td>
+                                         <td>3 \(\mu\)L</td>
-                                         <td>2 \(\mu\)l</td>
+                                         <td>2 \(\mu\)L</td>
-                                         <td>1 \(\mu\)l</td>
+                                         <td>1 \(\mu\)L</td>
                                  </tr>
                              </tbody>
@@ Line 749: / Line 788: @@
                  <br/>
                  <hr/>
-                 <div id="section-5-2" class="subgroup">
+                 <div id="section-5-2 " class="subgroup ">
                      <h4>5.2 Transformation</h4>
                      <ul>
@@ Line 755: / Line 794: @@
                              <h5>Material:
                                  </h5></li>
-                         <table class="table table-condensed">
+                         <table class="table table-condensed ">
                              <tbody>
                                  <tr>
@@ Line 773: / Line 812: @@
                                      <td>10 g/L
                                          <td>5 g/L</td>
-                                         <td>10 \(\mu\)l</td>
+                                         <td>10 \(\mu\)L</td>
                                  </tr>
                              </tbody>
@@ Line 781: / Line 820: @@
                              <h5>Protocol:</h5>
                              <p>preparation of the competent cells</p>
-                             <p>20\(\mu\)l ligation product + 50\(\mu\)l cells
+                             <p>20\(\mu\)L ligation product + 50\(\mu\)L cells
                              </p>
-                             <p>Heatshock of E.coli BW25113(42&#176C,90s)
+                             <p>Heatshock of E.coli BW25113(42&#176;C,90s)
                              </p>
                              <p>Put on ice(2min)
                              </p>
-                             <p>Add 800\(\mu\)l LB media and incubate for 1.5h(37&#176C, 150rpm)
+                             <p>Add 800\(\mu\)L LB media and incubate for 1.5h(37&#176;C, 150rpm)
                              </p>
-                             <p>Centrifuge at 4000rpm for 1min and remove 900\(\mu\)l supernatant
+                             <p>Centrifuge at 4000rpm for 1min and remove 900\(\mu\)L supernatant
                              </p>
                              <p>Resuspend the pellets using the left supernatant
@@ Line 795: / Line 834: @@
                              <p>Spread plates(with ampicillin)
                              </p>
-                             <p>Incubate for 12~16h(37&#176C)
+                             <p>Incubate for 12~16h(37&#176;C)
                              </p>
                          </li>
                      </ul>
                  </div>
-                 <div id="section-5-3" class="subgroup">
+                 <div id="section-5-3 " class="subgroup ">
                      <br/>
                      <hr/>
@@ Line 810: / Line 849: @@
                          <li>
                              <h5>Materials:</h5>
-                             <table class="table table-condensed">
+                             <table class="table table-condensed ">
                                  <tbody>
                                      <tr>
@@ Line 827: / Line 866: @@
                                      <tr>
                                          <td>Stacking Gel(4%) </td>
-                                         <td>pH=6.8 500\(\mu\)l
+                                         <td>pH=6.8 500\(\mu\)L
                                          </td>
-                                         <td>500 \(\mu\)l</td>
+                                         <td>500 \(\mu\)L</td>
-                                         <td>25 \(\mu\)l</td>
+                                         <td>25 \(\mu\)L</td>
-                                         <td>1350 \(\mu\)l</td>
+                                         <td>1350 \(\mu\)L</td>
-                                         <td>2.5 \(\mu\)l</td>
+                                         <td>2.5 \(\mu\)L</td>
-                                         <td>12.5 \(\mu\)l</td>
+                                         <td>12.5 \(\mu\)L</td>
                                      </tr>
                                      <tr>
                                          <td>Running Gel(12%)
-                                             <td>pH=8.8 1250 \(\mu\)l</td>
+                                             <td>pH=8.8 1250 \(\mu\)L</td>
-                                             <td>2000 \(\mu\)l</td>
+                                             <td>2000 \(\mu\)L</td>
-                                             <td>50 \(\mu\)l</td>
+                                             <td>50 \(\mu\)L</td>
-                                             <td>1675 \(\mu\)l</td>
+                                             <td>1675 \(\mu\)L</td>
-                                             <td>2.5 \(\mu\)l</td>
+                                             <td>2.5 \(\mu\)L</td>
-                                             <td>25 \(\mu\)l</td>
+                                             <td>25 \(\mu\)L</td>
                                      </tr>
                                      <tr>
                                          <td>Running Gel(18%)
-                                             <td>pH=8.8 1250 \(\mu\)l</td>
+                                             <td>pH=8.8 1250 \(\mu\)L</td>
-                                             <td>3000 \(\mu\)l</td>
+                                             <td>3000 \(\mu\)L</td>
-                                             <td>50 \(\mu\)l</td>
+                                             <td>50 \(\mu\)L</td>
-                                             <td>675 \(\mu\)l</td>
+                                             <td>675 \(\mu\)L</td>
-                                             <td>2.5 \(\mu\)l</td>
+                                             <td>2.5 \(\mu\)L</td>
-                                             <td>25 \(\mu\)l</td>
+                                             <td>25 \(\mu\)L</td>
                                      </tr>
                                  </tbody>
                              </table>
-                             <table class="table table-condensed">
+                             <table class="table table-condensed ">
                                  <tbody>
                                      <tr>
@@ Line 913: / Line 952: @@
                              <p>Divide the NGM solid medium (d = 6 cm) equally into two parts (drawing on the surface of the culture dishes).
                              </p>
-                             <p>Put 50 \(\mu\)l compound of different concentration and DMSO as contrast respectively on the two parts. Make 3 repeats of each concentration.
+                             <p>Put 50 \(\mu\)L compound of different concentration and DMSO as contrast respectively on the two parts. Make 3 repeats of each concentration.
                              </p>
-                             <p>Flush nematode from the plate with M9 and inoculate 20 \(\mu\)l (about 30 nematodes) into the center of the medium.
+                             <p>Flush nematode from the plate with M9 and inoculate 20 \(\mu\)L (about 30 nematodes) into the center of the medium.
                              </p>
-                             <p>Incubate at 20&#176C for 1hr, and then place them at 4&#176C refrigerator for 1hr.
+                             <p>Incubate at 20&#176;C for 1hr, and then place them at 4&#176;C refrigerator for 1hr.
                              </p>
                              <p>Count the nematodes of each part under the stereoscope.
@@ Line 930: / Line 969: @@
                      <ul>
-                         <p>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&#176C, 190r.m.p for 3 hr.
+                         <p>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&#176;C, 190 r.m.p for 3 hr.
                          </p>
-                         <p>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&#176C overnight.
+                         <p>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&#176;C overnight.
                          </p>
-                         <p>Flush nematodes from the plate with M9, centrifugate (1500r.p.m,3min), abandon the supernatant and resuspend the precipitation with 3mL M9.
+                         <p>Flush nematodes from the plate with M9, centrifugate (1500 r.p.m, 3min), abandon the supernatant and resuspend the precipitation with 3mL M9.
                          </p>
-                         <p>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&#176C incubate overnight.
+                         <p>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&#176;C incubate overnight.
                          </p>
                          <p>Observe and compare the activation and size of nematodes of each group and draw a conclusion.
@@ Line 944: / Line 983: @@
                  <br/>
                  <hr/>
-                 <div id="section-5-4" class="subgroup">
+                 <div id="section-5-4 " class="subgroup ">
                      <h4>5.4 Nematode</h4>
                      <ul>
@@ Line 953: / Line 992: @@
                              <p>NGM medium:</p>
                          </li>
-                         <table class="table table-condensed">
+                         <table class="table table-condensed ">
                              <tbody>
                                  <tr>
@@ Line 973: / Line 1,012: @@
                                      <td>3 g
                                          <td>2.5 g/L</td>
-                                         <td>17 \(\mu\)l</td>
+                                         <td>17 \(\mu\)L</td>
-                                         <td>975 ml</td>
+                                         <td>975 mL</td>
                                  </tr>
                              </tbody>
@@ Line 980: / Line 1,019: @@
                          <p>Prepare 1mol/L CaCl<sub>2</sub>, 1mol/L MgSO<sub>4</sub> and phosphoric acid buffer meanwhile.
                          </p>
-                         <table class="table table-condensed">
+                         <table class="table table-condensed ">
                              <tbody>
                                  <tr>
@@ Line 1,003: / Line 1,042: @@
                              </tbody>
                          </table>
-                         <p>Autoclave the above mentioned reagents with 120&#176C for 30 min and then water bath them to 65&#176C.</p>
+                         <p>Autoclave the above mentioned reagents with 120&#176;C for 30 min and then water bath them to 65&#176;C.</p>
-                         <p>Dissolve the cholesterol with ethanol to 5 mg/ml.</p>
+                         <p>Dissolve the cholesterol with ethanol to 5 mg/mL.</p>
-                         <p>Add 1ml cholesterol, CaCl<sub>2</sub>, MgSO<sub>4</sub> and 25 mL phosphoric acid buffer into the minimum medium in order (all at 65&#176C, shake up).</p>
+                         <p>Add 1ml cholesterol, CaCl<sub>2</sub>, MgSO<sub>4</sub> and 25 mL phosphoric acid buffer into the minimum medium in order (all at 65&#176;C, shake up).</p>
-                         <p>Make plate with the NGM medium, storing at 4&#176C.</p>
+                         <p>Make plate with the NGM medium, storing at 4&#176;C.</p>
                          <br/>
                          <li>
                              <h5>OP50 E.coli deficit type (food of nematodes):</h5></li>
-                         <p>Streak inoculate OP50 on LB solid medium, Incubate at 37&#176C for 6 hr or overnight. Inoculate LB liquid medium with OP50 single colonies, and incubate (37&#176C, 220r.p.m) overnight.
+                         <p>Streak inoculate OP50 on LB solid medium, Incubate at 37&#176;C for 6 hr or overnight. Inoculate LB liquid medium with OP50 single colonies, and incubate (37&#176;C, 220 r.p.m) overnight.
                          </p>
                      </ul>
@@ Line 1,020: / Line 1,059: @@
                              <h5>The incubation condition of caenorhabditis elegans N2 wild type:
 </h5>
-                             <p>Generally incubated at 20&#176C. Grow slowly at 16&#176C and grow fast at 25&#176C while egg laying amount declines.</p>
+                             <p>Generally incubated at 20&#176;C. Grow slowly at 16&#176;C and grow fast at 25&#176;C while egg laying amount declines.</p>
                          </li>
                          <br/>
@@ Line 1,026: / Line 1,065: @@
                              <h5>Protocol:
                                  </h5></li>
-                         <p>Inoculate NGM medium with 150~200 \(\mu\)l OP50, incubate at 37&#176C for 12hr. Cut down a square of NGM(about 1cm x 1cm) with nematodes.
+                         <p>Inoculate NGM medium with 150~200 \(\mu\)L OP50, incubate at 37&#176;C for 12hr. Cut down a square of NGM(about 1cm x 1cm) with nematodes.
                          </p>
                          <p>Put the square on the NGM medium with OP50 and let the surface with nematodes adown to contact the medium.
                          </p>
-                         <p>Seal and incubate upside down at 20&#176C.
+                         <p>Seal and incubate upside down at 20&#176;C.
                          </p>
                          <p>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.
                          </p>
-                         <p>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 1500r.m.p for 1 min, and incubate the precipitate on a new plate. </p>
+                         <p>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. </p>
                          <br/>
                          <h5 align='center'>Synchronization</h5>
@@ Line 1,047: / Line 1,086: @@
                          </li>
-                         <table class="table table-condensed">
+                         <table class="table table-condensed ">
                              <tbody>
                                  <tr>
@@ Line 1,071: / Line 1,110: @@
                                          <td>3 g</td>
                                          <td>5 g</td>
-                                         <td>1 ml</td>
+                                         <td>1 mL</td>
                                          <td>1 L
                                          </td>
@@ Line 1,078: / Line 1,117: @@
                          </table>
-                         <table class="table table-condensed">
+                         <table class="table table-condensed ">
                              <tbody>
                                  <tr>
@@ Line 1,095: / Line 1,134: @@
                                  <tr>
                                      <td>Dosage</td>
-                                     <td>50 \(\mu\)l
+                                     <td>50 \(\mu\)L
-                                         <td>100 \(\mu\)l</td>
+                                         <td>100 \(\mu\)L</td>
-                                         <td>850 \(\mu\)l</td>
+                                         <td>850 \(\mu\)L</td>
                                  </tr>
                              </tbody>
@@ Line 1,110: / Line 1,149: @@
                          <p>Flush the nematodes with M9 NS
                          </p>
-                         <p>Centrifugation (3000r.m.p, 1min)
+                         <p>Centrifugation (3000 r.m.p, 1min)
                          </p>
-                         <p>Abandon the supernatant, add 1mL bleach buffer to the precipitation
+                         <p>Abandon the supernatant, add 1 mL bleach buffer to the precipitation
                          </p>
-                         <p>Centrifugation (3000r.m.p, 1min), abandon the supernatant
+                         <p>Centrifugation (3000 r.m.p, 1min), abandon the supernatant
                          </p>
                          <p>Transfer the precipitation (nematode eggs) to a NGM plate with OP50, then the eggs will incubate synchronously.
@@ Line 1,123: / Line 1,162: @@
                          </p>
                          <p>
-                             Incubate the plate at 20&#176C for 3d and therefor can get about 300 to 400 nematodes.
+                             Incubate the plate at 20&#176;C for 3d and therefor can get about 300 to 400 nematodes.
                          </p>
                          <p><strong>Note: all of the procedure outlined above must be conducted under sterile condition.
@@ Line 1,132: / Line 1,171: @@
                          <li>
                              <h5>modified Baermann funnel method:</h5></li>
-                         <p> 1. Place a glass funnel (d = 10~15cm) on the funnel stand linked with about 10cm rubber tube which is furnished with a flatjaw pinchcock.
+                         <p> 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.
                          </p>
-                         <p>2. Cut the soil sample into matchstick shape. Package 10g(wet weight) with gauze and put it into the funnel, and then add water to immersion the sample.
+                         <p>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.
                          </p>
                          <p>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.
@@ Line 1,146: / Line 1,185: @@
      </div>
-        <footer>
+<footer>
-            <div class="container">
+        <div class="container ">
-                <div class="col-md-9 text-center col-md-offset-3"><img src="https://static.igem.org/mediawiki/2015/c/c3/BANNER.png" alt="igem banner" /></div>
+            <div class="col-md-9 text-center col-md-offset-3 "><img src="img/igem-bannner.png " alt="igem banner " /></div>
-            </div>
+        </div>
          </footer>

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

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

Difference between revisions of "Team:BNU-CHINA/Team"

Revision as of 04:11, 14 September 2015

1. Attribution

Project Design and Wet Lab Experiments

Modeling

Wiki Construction

Graphic design

Human practice

2. Collaboration

3. Acknowledgement

Thanks:

Prof. Zhu Xudong, Prof. Yang Dong and Prof. Xiang Benqiong

Hao Xiaoran, Chi Xiaodong, Huo Liang, Ai Ying and Wang Xuan

The College of Chemistry, Beijing Normal University

Prof. Yang Chonglin of Chinese Academy of Sciences

Prof. Li Hongmei of Department of Plant Pathology，Nanjing Agricultural University

BIT-China & ZJU-China & FAFU-CHINA

Agricultural Service Center of Ceyu, Hebei Province

Shang Zhong National Middle School of Guizhou Province

National Chiao Tung University &Peking University

Feng Xiaofei and her family in Hebei Province

Prof. Wang Yingdian

Sponsors:

4. Notebook

5.1 Cloning

PCR

Reaction System:

Process:

Electrophoresis---Gel Purification

Material:

Protocol:

Digestion

Ligation

5.2 Transformation

Material:

Protocol:

5.3 Detetion

SDS-PAGE

Materials:

Protocol:

Detection of Baiting Nematodes

Materials:

Protocol:

Detection of Killing Nematodes

5.4 Nematode

Materials:

OP50 E.coli deficit type (food of nematodes):

Cultivate

The incubation condition of caenorhabditis elegans N2 wild type:

Protocol:

Synchronization

Materials:

Protocols:

Isolate nematodes from soil

modified Baermann funnel method: