Team:Jilin China/Notebook

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Notebook

We started our experiments in May. Before that, we read many related articles and planned our experiments. After that, we found out the sequence that we needed and had the first plasmid synthesized.

Week 1 (May 9-May 15)

We prepared the LB medium, and transform the plasmid into DH5α. After there were some colonies, we picked the monoclonal colony and purified plasmid. However, the concentration of plasmid was not high enough.

Week 2 (May 16-May 22)

We repeated the same experiment as last week. We transformed the plasmid into BL21 and induced the expression of the apoptin by IPTG at 37℃ for 4 hours.

Week 3 (May 23-May 29)

We did the SDS-PAGE by the samples that made and boiled on the last week. However, the result was not the same as we expected. We did not see the band at the expected size of 13.6Kd. So, we did the SDS-PAGE again, but the result was also not as we expected.

Notebook

Week 4 (May 30-June 5)

We transformed the plasmid into BL21 and induced the expression of the apoptin by IPTG at 28℃ overnight. We did the SDS-PAGE by the samples. However, the result was not the same as we expected. So, we decided to improve it. We sonicated the bacteria, and used the supernatant to do the SDS-PAGE. However, we did not get the result as we expected. We prepared PYG medium and activated freeze-drying bifidobacterium longum(bifi) and passaged them everyday.

Notebook

Week 5 (June 6-June 12)

We transformed the plasmid into BL21 and induced the expression of the apoptin by IPTG at 28℃ overnight. We sonicated the bacteria, and we used the supernatant for SDS-PAGE. We could see the band of apoptin at the expected molecular weight this time. So, we did the western blot to prove the expression of apoptin. However, the result was not the same as we expected. We continued to passage and keep the bifi.

Notebook

Week 6 (June 13-June 19)

We sonicated the bacteria, and used the supernatant to do the western blot to prove the expression of apoptin. We could see the band of apoptin this time. We also transform the plasmid into DH5α. After there were some colonies, we picked the monoclonal colony and purified plasmid. We made the competent Bifi cell and transferred the methylated plasmid into the competent Bifi cell by electro transformation. The result showed the electro transformation was successful. Then we picked the monoclonal colony and cultured it to make the samples of SDS-PAGE and western blot.

Week 7 (June 20-June 26)

We did the SDS-PAGE and western blot by the samples that we made last week. However, the result was not same as we expected. So, we did it again from the electro transformation to western blot. We could see the band of apoptin. At the same time, we transfect the plasmid into the MCF-7 cell to prove that apoptin can be harmful to solid tumor cells. We also did the MTT experiment and wound scratch healing assay to prove the function of apoptin. The results were very good. Because of the final examinations, we paused our experiments for 2 weeks.

Notebook

Week 10 (July 10-July17)

We read many articles to improve our project. We add signal peptides (Tmp1 and Sec2) to help the apoptin secrete from the bifi and TAT to help the apoptin enter into the cells. We designed three plasmids and had them synthesized them by company. We got the plasmids after 28 days.

Week 14 (August 15-August 21)

We transform the three new plasmids into DH5α.After there were some colonies, we picked the monoclonal colonies and purified plasmids. We transferred the plasmids into the competent Bifi cell by electro transformation. However, the electro transformation was failed, since there were no colonies on the BBL medium. We tried it again, but the result was not good. The plate of control plasmid showed some colonies, but the experimental group did not grow any colony.

Notebook

Week 15 (August 22-August 28)

We made some new competent Bifi cells and transferred the plasmids into the competent Bifi cell by electro transformation. However, the result was not good. The plate of control grow some colonies, but the experimental group did not show any colonies. We tried the electro transformation twice this week, but the results were not good.

Week 16 (August 29-September 4)

We tried the electro transformation again, but the result was not good. So, we decided to change something. We made some new competent Bifi cells again. We also modified the condition of the electro transformation (changed the voltage from 1800V to 1250V,1500V and 2000V). The result showed the electro transformation was successful, there were some colonies on the plates. We picked the monoclonal colonies into the PYG medium to culture them. We treated them in centrifuge (8000rmp, 10 minutes) and collected the supernatant to make the sample for SDS-PAGE. The pellet was sonicated with 7 second work and 30 second pulse in ice for 20 minutes. After centrifugation (4℃,12000rmp,20min) , the supernatant was collected into 1.5ml EP tubes to make samples for SDS-PAGE. The pellet was used to make samples for SDS-PAGE and marked. We did SDS-PAGE and Western Blot by the samples.

Notebook

Week 17(September 5-September 11)

We made the lyophilized powder by skim milk powder to keep the bifi that transformed the plasmids. We counted the number of active bacteria. We ordered some mice to do the animal experiment.

Week19 (September 16-September 26)

We received the mice and kept them for 10 days to adapt to the environment of our laboratory. At the same time, we transform the three new plasmids into BL21 to test the expression of the new protein in E.coil. We used the sample to do the SDS-PAGE and Western Blot.

We started our animal experiment at September 26.

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