Team Notebook
![](https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png)
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.
![](https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png)
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!
![](https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png)
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.
![](https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png)
Modeling group members read some related materials and papers, including wikis of former teams especially their modeling parts as well as papers introducing nematodes.
![](https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png)
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.
![](https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png)
![](https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png)
![](https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png)
![](https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png)
![](https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png)
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.
![](https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png)
![](https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png)
![](https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png)
![](https://static.igem.org/mediawiki/2015/2/25/BNU-bace16.png)
![](https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png)
We did restriction enzyme digestion to check the extracted plasmid.
![](https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png)
![](https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png)
![](https://static.igem.org/mediawiki/2015/2/25/BNU-bace16.png)
![](https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png)
![](https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png)
![](https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png)
![](https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png)
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.
![](https://static.igem.org/mediawiki/2015/2/25/BNU-bace16.png)
![](https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png)
We cultivated nematodes.
![](https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png)
![](https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png)
![](https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png)
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).
![](https://static.igem.org/mediawiki/2015/2/25/BNU-bace16.png)
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 |
![](https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png)
![](https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png)
![](https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png)
![](https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png)
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.
![](https://static.igem.org/mediawiki/2015/2/25/BNU-bace16.png)
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.
![](https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png)
![](https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png)
![](https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png)
![](https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png)
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.
![](https://static.igem.org/mediawiki/2015/2/25/BNU-bace16.png)
![](https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png)
![](https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png)
![](https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png)
![](https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png)
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.
![](https://static.igem.org/mediawiki/2015/2/25/BNU-bace16.png)
![](https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png)
We prepared NGM plate and nematodes liquid medium for toxic test of rMpL.
![](https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png)
![](https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png)
![](https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png)
We re-prepared the homogenate of the revivedE.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.
![](https://static.igem.org/mediawiki/2015/2/25/BNU-bace16.png)
No Bace16 protein are found in supernatant.
![](https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png)
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.
![](https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png)
![](https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png)
![](https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png)
![](https://static.igem.org/mediawiki/2015/2/25/BNU-bace16.png)
![](https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png)
Run SDS-PAGE and get no positive results.
Cultivate recombinant bacteria, bacteria with empty vector or OP50 bacteria for nematoxicity test of rMpL protein.
![](https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png)
![](https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png)
![](https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png)
![](https://static.igem.org/mediawiki/2015/2/25/BNU-bace16.png)
![](https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png)
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.
![](https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png)
![](https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png)
![](https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png)
![](https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png)