Difference between revisions of "Team:ZJU-China/Results"
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<p> | <p> | ||
The mamC-ZZ could bind secondary antibody by Fc region directly and the band would appear at 26 kDa approximately. Lane 2 indicated that scFv-Fc was immobilized on the resin, | The mamC-ZZ could bind secondary antibody by Fc region directly and the band would appear at 26 kDa approximately. Lane 2 indicated that scFv-Fc was immobilized on the resin, | ||
| − | meanwhile, the ZZ region specifically interacted with the Fc region and also showed a band ( | + | meanwhile, the ZZ region specifically interacted with the Fc region and also showed a band (Figure 8A). Furthermore, lane 3 suggested a more inspiring result, which meant |
scFv-Fc could bind mamC-ZZ in a complicated environment. These results implied us an easier way to purified and enriched mamC-ZZ from cell lysate directly. | scFv-Fc could bind mamC-ZZ in a complicated environment. These results implied us an easier way to purified and enriched mamC-ZZ from cell lysate directly. | ||
</p> | </p> | ||
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indicates clearly that scFv can specifically bind to HER2 on tumor cell membrane (Figure 10D). | indicates clearly that scFv can specifically bind to HER2 on tumor cell membrane (Figure 10D). | ||
</p> | </p> | ||
| + | <br> | ||
| + | <br> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="section about" id="expression_of_mamC_ZZ"> | ||
| + | <div class="container1"> | ||
| + | |||
| + | <h2><b>Culture of Magnetotactic Bacteria</b></h2> | ||
| + | <br> | ||
| + | <br> | ||
| + | <h2>Microaerobic Culture</h2> | ||
| + | <p> | ||
| + | As magnetotactic bacteria needed microaerobic conditions to grow, we chose liquid medium rather than solid for the growth of bacteria. In order to show the change of oxygen content in the culture medium visibly, we added resazurin into the medium. Resazurin is an indicator of dissolved oxygen. It turns blue when there is dissolved oxygen in the solution and turns red when there is no oxygen. The results showed that the color of the liquid medium which were added into MSR-1 were obviously different from that without the addition of MSR-1 (Figure 11), indicating that the dissolved oxygen may be consumed out by MSR-1. | ||
| + | </p> | ||
| + | <div class="imgbox"> | ||
| + | <img src="https://static.igem.org/mediawiki/2020/9/95/T--ZJU-China--MSR-1.jpeg" alt=""> | ||
| + | <h6>Figure 11. Liquid medium cultured MSR-1 (red test tubes) contrast to the blank control (blue test tubes).</h6> | ||
| + | </div> | ||
| + | <br> | ||
| + | <br> | ||
| + | <h2>Bacterial PCR of MSR-1</h2> | ||
| + | <p> | ||
| + | To further demonstrate that the magnetotactic bacteria was truly grown in the liquid medium, bacterial PCR was carried out using specific primers designed from the 16srDNA of <i>M. gryphiswaldense</i> and used <i>E.coli</i> and <i>Agrobacterium</i> as the negative control. As the results of bacterial PCR showed, there was a bright band in lane2, while no bands in lane 3 and lane 4, demonstrating that MSR-1 was successfully grown up (Figure 12). | ||
| + | </p> | ||
| + | <div class="imgbox"> | ||
| + | <img src="https://static.igem.org/mediawiki/2020/1/19/T--ZJU-China--bacterial_PCR_of_MSR-1.jpeg" alt=""> | ||
| + | <h6>Figure 12. Results of the bacterial PCR.</h6> | ||
| + | <br> | ||
| + | <br> | ||
| + | </div> | ||
| + | |||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 04:48, 27 October 2020
Results
Expression of scFv-Fc
If not specified, all expression chassis of scFv is SHuffle®. The supernatant and the pellet of cell lysate was analyzed by SDS–PAGE, corresponding to lane 1 and lane 2, respectively. According to the Coomassie staining result below, we pointed out that scFv-Fc was presented in the correct weight and expression level was higher in supernatant rather than pellet, indicating the solubility of this fusion protein.
Figure 1. Coomassie staining results of scFv-Fc to investigate the solubility.
Besides, scFv-Fc was also expressed in BL21 (DE3), which meant the absence of two disulfide bonds, leading to the incorrect folding of scFv. Therefore, the protein expressed in BL21 (DE3) cannot interact with HER2 theoretically.
Optimum Concentration of IPTG for Expression
We carried out a gradient experiment to investigate the optimum concentration of IPTG for expressing. The transformed cells were grown until OD600 reached 0.6, we introduced the expression with IPTG, to a final concentration of 0.5, 1, and 2 mM, respectively. For negative control, IPTG was not introduced.
Figure 2. Western blotting results of scFv-Fc under different inducing concentration of IPTG.
As Figure 2 shows above, the best concentration for expressing was 2 mM of IPTG. There was a small amount of expression without IPTG induction, probably due to promoter leakage. The expression level of IPTG decreased first and then increased with the increase of IPTG concentration. This may cause by the inhibitory effect of IPTG on bacterial growth.
Optimum Induction Time for Expression
A gradient experiment was performed to make sure that the target protein was induced and to figure out the best time for expressing. When OD600 reached 0.6, 2 mM of IPTG was induced. The expression time was 0, 1, 8, and 24 h, respectively. For negative control, none inducer was introduced.
Figure 3. Western blotting results of scFv-Fc under different induction time.
As Figure 3 shows above, the longer time for expressing, the higher expression level the cells reached. In the next work, the expression period was carried out under 2 mM of IPTG inducing and 24 h of additional incubating.
Purification of the Proteins
We perform the immunoprecipitation (IP) to obtain purified FLAG-tagged protein. IP has been described in detail in the experiment section. The target protein appeared at 54kDa. Nevertheless, there were a lot of proteins in the high molecular weight part, because scFv would form a polymer, which was immobilized as well. At the same time, there was protein in small the molecular weight part, which may be caused by the protein truncating easily at the junction.
Figure 4. Western blotting results of immunoprecipitation of scFv-Fc.
Expression of mamC-ZZ
Optimum Concentration of IPTG for Expression
A gradient experiment was conducted to investigate the optimum IPTG concentration for GST mamC-ZZ expression. The transformed bacteria were cultured until OD600 reached 0.6, we induced the expression with IPTG, to a final concentration of 1, 2, and 3mM, respectively. For negative control, IPTG was not introduced.
Figure 5. Western blot results of GST mamC-ZZ under different inducing IPTG concentration. As results shown above, the best concentration for expressing was 2 mM of IPTG.
As Figure 5 is shown above, the best concentration for expressing was 2 mM of IPTG.
Optimum Inducion Iime for Expression
A gradient experiment was carried out to investigate the optimum inducing time for GST mamC-ZZ expression. The transformed bacteria were cultured until OD600 reached 0.6 and induced the expression with IPTG. In a single experimental blocks, a group of gradient expressing time, which set from 1 h to 4 h, was tested.
Figure 6. Western blot results of GST mamC-ZZ under different induction time.
Purification of the Proteins
Immunoprecipitation (IP) was performed to obtain purified GST-tagged protein and investigate the optimum condition of purification. IP has been described in detail in the experiment section. The target protein appeared at 52kDa. A group of gradient glutathione resin, which added as single, double and triple volume of GST mamC-ZZ solution, was tested.
Figure 7. Coomassie staining results of GST mamC-ZZ under different purification conditions
As results are shown above, the efficiency of purification did not demonstrate a significant difference among different glutathione resin volumes which added in. In the following work, we added equal glutathione resin as GST mamC-ZZ solution to purify the recombinant protein.
Protein Interaction
Immunoprecipitation
To prove that mamC-ZZ can combine with scFv-Fc, we conducted the immunoprecipitation experiments.
Lane 1 stands for input, which was loaded with cell lysate of which obtained mamC-ZZ and purified. Lane 2 was loaded with IP products of cell lysate of which obtained mamC-ZZ
and purified. Lane 3 was loaded with IP products of purified mamC-ZZ and scFv. Lastly, lane 4 was loaded only with scFv. All the samples were digested by thrombin to cut off
any potential GST region.
Figure 8. Western blot results of mamC-ZZ and scFv which introduce different primary antibody. One block was incubated with rabbit anti-DDDDK tag (binds to FLAG® tag sequence) antibody and goat anti-rabbit IgG H&L (HRP), and another block was only incubated with goat anti-rabbit IgG H&L (HRP), which can interact with mamC-ZZ by Fc region and shows the specific position of mamC-ZZ; B. Western-blot results of the input control block.
The mamC-ZZ could bind secondary antibody by Fc region directly and the band would appear at 26 kDa approximately. Lane 2 indicated that scFv-Fc was immobilized on the resin, meanwhile, the ZZ region specifically interacted with the Fc region and also showed a band (Figure 8A). Furthermore, lane 3 suggested a more inspiring result, which meant scFv-Fc could bind mamC-ZZ in a complicated environment. These results implied us an easier way to purified and enriched mamC-ZZ from cell lysate directly.
Specifically Target HER2
Cultivation of Tumor Cells
We cultured two kinds of tumor cells which is MDA-MB-253 represented HER2 positive breast cancer and MDA-MB-231 represented HER2 negative breast cancer.
Figure 9. Images of MDA-MB-253 and MDA-MB-453 at different time after subculture. (A). This image of MDA-MB-231 was taken one day after subculturing, the amount of cell is low; (B).This image of MDA-MB-231 was taken four days after subculturing, tumor cell was at good survival conditions and developed a high cell intensity; (C).This image of MDA-MB-453 in medium intensity was taken at six days after subculturing; (D).Ten days after subculturing, MDA-MB-453 showed a great cell intensity, and the morphology of some tumor cells has transformed account for long-time cultivation.
As images are shown above, both MDA-MB-453 and MDA-MB-231 was cultured successfully and prepared to attend further experiments steps.
Flow Cytometry
MDA-MB-453 and MDA-MB-231 was both incubated with scFv-Fc on ice for 30 minutes, and stained with Fixable Viability Dye eFluor 450 and Alexa Fluor 488 for flow cytometry.
Figure 10. Flow cytometry results of MDA-MB-453 and MDA-MB-231 after incubated with scFv-Fc.
As results are shown above, the fluorescence intensity of MDA-MB-453 cells which incubated with scFv-Fc was significantly higher than the other MDA-MB-453 cells from the negative
control group, which indicated that scFv-Fc could specifically bind with the certain target on MDA-MB-453 membrane (Figure 10A).
At the same time, we can also see that MDA-MB-231 has an obvious fluorescence peak after incubated by scFv-Fc (Figure 10B).
Then, the following analysis shows that although scFc-Fc can target both MDA-MB-453 and MDA-MB-231 cells, the fluorescence peaks were significantly different. Obviously, the
high HER2 expression cell line (MDA-MB-453) showed a higher fluorescence than that of the low HER2 expression cell line (MDA-MB-231), indicating that scFv-Fc is more targeted
to HER2, and can distinguish breast cancer cells with high and low expression of HER2 (Figure 10C). And the fluorescence of MDA-MB-453 was about 10 times higher than
MDA-MB-231’s, which was corresponding with the difference of HER2 expression level between HER2 positive and negative cells mentioned in existing studies[1].
To put it more bluntly, compared with MDA-MB-231 which is HER2-negative, MDA-MB-453 had significantly higher fluorescence intensity when both cells were alive. This results
indicates clearly that scFv can specifically bind to HER2 on tumor cell membrane (Figure 10D).
Culture of Magnetotactic Bacteria
Microaerobic Culture
As magnetotactic bacteria needed microaerobic conditions to grow, we chose liquid medium rather than solid for the growth of bacteria. In order to show the change of oxygen content in the culture medium visibly, we added resazurin into the medium. Resazurin is an indicator of dissolved oxygen. It turns blue when there is dissolved oxygen in the solution and turns red when there is no oxygen. The results showed that the color of the liquid medium which were added into MSR-1 were obviously different from that without the addition of MSR-1 (Figure 11), indicating that the dissolved oxygen may be consumed out by MSR-1.
Figure 11. Liquid medium cultured MSR-1 (red test tubes) contrast to the blank control (blue test tubes).
Bacterial PCR of MSR-1
To further demonstrate that the magnetotactic bacteria was truly grown in the liquid medium, bacterial PCR was carried out using specific primers designed from the 16srDNA of M. gryphiswaldense and used E.coli and Agrobacterium as the negative control. As the results of bacterial PCR showed, there was a bright band in lane2, while no bands in lane 3 and lane 4, demonstrating that MSR-1 was successfully grown up (Figure 12).
Figure 12. Results of the bacterial PCR.
References
[1]. Press, M. F., Pike, M. C., Chazin, V. R., Hung, G., Udove, J. A., Markowicz, M., Danyluk, J., Godolphin, W., Sliwkowski, M., & Akita, R. (1993). Her-2/neu expression in node-negative breast cancer: direct tissue quantitation by computerized image analysis and association of overexpression with increased risk of recurrent disease. Cancer research, 53(20), 4960–4970.