Team Notebook
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!
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.
There are some problems of rMpL and bace16 gene synthesis. GENEWIZ gave us the relevant feedback in time and modified the two sequences to synthesize again.
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.
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.
We designed the sequence of Limonene synthase (LS) that would be transformed to E. coli to express. Our designed sequences were 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.
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 bacteria 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 bacteria 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 regarded nematodes as static models couldn't 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.
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 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 could 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 environment-friendly device to kill nematodes. Finally our plan is to design and build a debug device.
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 doing 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 satisfying.
| 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 is 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 hands.
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 -20°, 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.
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 powder. We dissolved these dry powder 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 were obvious when ran SDS-PAGE together whereas the objective band of BW25113 couldn't 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 bacteria, 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 could 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.
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 correct. And we sent 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-pSB1c3) 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 controll 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 plates 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.
We transferred plasmids with correct sequenced d&l-limonene synthase and GPPS gene into expression strain E. coli BL21 respectively to express LS and GPPS. We also transferred empty pSB1C3 plasmids into expression strain E. coli BL21 and regarded them as the control group. After being amplification cultured and expressing aiming products, the bacteria were smashed by ultrasonication. We tested whether the protein expressed or not by SDS-PAGE for homogenate and supernatant. The results of SDS-PAGE suggested that d&l-limonene synthase and GPPS were expressed successfully.
Incubate the bacteria with 8 \(\mu\)M, 10 \(\mu\)M and 12 \(\mu\)M arabinose , run SDS-PAGE after ammonium sulfate precipitation.
No Bace16 protein are found in supernatant.
Change the E. coli strain to express rMpL, only add 10 \(\mu\)M and 12 \(\mu\)M arabinose for incubation this time.
To test if rMpL is expressed in the pallet or the supernatant, ammonium sulfate precipitation is used to enrich the protein.using Ultrasonication and SDS-PAGE are also used during this process.
This week we discussed factors that would influence the putting model of equipment on the farmland. Considering there are so many factors can’t be predicted in the real situation, we talked about how to idealize and simplify our putting model.
In order to testify if engineering bacteria have the ability to express limonene, amplification cultured bacteria liquid covered with n-hexane was cultured in the shaking table to express production. We used n-hexane to extract possible limonene from fermentation broth. Then we dried our samples by Na2SO4. We plan to use GC-MS to verify the existent of limonene. At the same time, we carried out the experiment of attractive interactions of limonene towards nematodes several times. The results verified that compound limonene could attract nematodes preliminarily.
Cultivate C. elegans larva after synchronization on the NGM medium with bacteria of experimental group and control group. Observe growth state of C.elegans every 12 hours. No positive results are observed in 4 days.
Ammonium sulfate precipitation.
Run SDS-PAGE but get no positive results.
Cultivate recombinant bacteria, bacteria with empty vector or OP50 bacteria for nematoxicity test of rMpL protein.
As we discussed the influencing factors last week, our main work this week was to search documents to see what exact influence these factors would bring to our result, and whether these factors can be ignored or idealized. Also we improved our device and made a 3D model of it, which we called Device 2.0.
To verify whether limonene had been synthesized by bacteria, the fermentation broth was tested by GC-MS in School of Chemistry, Beijing Normal University. Results suggested that there was d-limonene in fermentation. It proved that limonene had been synthesized by engineering bacteria. The verification experiments of attractive interactions of limonene towards nematodes continued. We repeated verification experiments and assayed the data from experiments. According to pair t test, we concluded that limonene could attract nematodes.
According to the original paper, rMpL is soluble protein expressed inside the cell, so target protein should be still in pallets. Run SDS-PAGE and get bands which might be rMpL.
Cultivate C. elegans larva after synchronization on the NGM medium with bacteria. Observe growth state of C. elegans every 12 hours. Inhibition is observed on the plate inoculated with recombinat bacteria.
Through a lot of discussion among our group, and with accumulating realization and knowledge of this program, we finally came up with a method to solve the problem of how to place equipment we made on the farmland. We made a video to show the structure of Device 2.0.
All data from our experiments were reduced and summarized. All results were uploaded to our wiki.
This week we mainly collect files we used and results we got, thus we finally start to write the paper illustrating our achievements. At the same time, we make the model revision.
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