Team:Nanjing China Bio/notebook6

6.1(all)
This weekend, we had our third regular meeting, and had a new teammate joining in us. Jiren firstly further explained to us the conceptions we didn't figure out last week. Chao, our team leader, introduced the fundamental knowledge about cancer therapies and the background of the review, combining with his own experience in previous experiments. He also presented the weakness of the anti-cancer drugs nowadays, and the orientation scientists are working towards, which made us have a deep understanding of our project.
Afterwards, Anqi, Xianwei, and Jingwei gave us their presentations on specific bacterial targeting of tumors, intratumoral penetration, and native bacterial cytotoxicity. An later added illustrations on Salmonella typhimurium with the information he found.
During the presentations, other students raised questions to further dig into the review. For example, when talking about TNF-α, we had doubts on human immune system, the infection of S. typhimurium, and the function of this factor. Though we couldn't explain all these questions now, these discussions indeed broadened our minds.
In the end of the meeting, we assigned everyone tasks as usual. Although the exams are coming, giving us pressure on our study, we will still working hard towards our goal.

6.9(all)
Today we continued to focus on document reading like last week.
Bomiao and Bei firstly illustrated on cytotoxic agents, cytokines, tumor-specific antigens and antibodies, gene transfer and gene silencing. Among all these topics, the one of expression of anti-cancer agents caught the attention of us all. Everyone presented his own opinions on this topic, especially in the transfer of genetic material in mammals by bacteria.
In the second part, Wenping described to us particularly on gene-triggering strategies, focusing on the pictures of the mechanism. She tried hard to explain in detail, and asked us to understand it with the knowledge of molecular biology. And it worked out.
After that, our new teammate Zhifei Yuan elaborated on detection. Though he doesn't major in biology and is new to our team, his thinking is distinct and in order, and prepared well in the background knowledge.
Finally, Xiang concluded on applications of bacteria in cancer therapies. Apart from the existing techniques and methods, and the development situation, he also gave us his prediction on the developments of bacteria targeting cancer therapy in the future. Through his presentation, we learned more about our orientation of work ahead.

6.16(A)
In this meeting, we talk about the experiments we fall.
Recently we tried to knock out the relevant genes of VNP20009. But there were no positive results. After second consideration, we make our next plan. In the future we are going to try different concentrations of L-arabinose, We will assay 1 mM (indicated in the original protocol), 10 mM and 0.1 M of L-arabinose. After transformation of the amplimer, the cells were recovered in 1 ml of LB and incubated for 1 hour at 37°C (as suggested in the original method). In the next week we will try a serious of time of incubation and temperature 30°C before plating.
We hope we can have a good result.
Journals of experiments A
2012.6.2
1. Design primers.
2. Amplify a kanamycin cassette from PKD4.
2012.6.3
1. Prepare the competent cells for electroporation.
2. Introduce PKD4 into VNP20009 using electroporation procedure.
2012.6.4
1. Pick up the single colony.
2. Grow the VNP20009 harboring the PKD4 in LB +AMP at 30℃ overnight.
2012.6.5
1. The kanamycin cassette with the relevant homologous region was successfully amplified by the PCR.
2. Purify the PCR DNA and store at -80℃.
3. Preserve the VNP20009 with PKD4 in 20% glycerol.
2012.6.6
1. Introduce the kanamycin cassette with the relevant homologous region into the VNP20009 with PKD4.
2012.6.7
1．No colony was found on the plate.
2012.6.9
1. Tremendous small colonies were found on the plate. However, none of them did grow in the LB+kna.
2. We didn't get the relevant mutants in VNP20009.
2012.6.10
1. Amplify the the kanamycin cassette with the relevant homologous region with PCR.
2. Purify the he PCR DNA with bocai kit and elute in 30L dd water.
3. Store at -80℃.
2012.6.11
1. Prepare the competent cells for electroporation. (The final concentration of the arabinose was 100M and the inducible time is 2 hours.)
2012.6.12
1. Introduce the kanamycin cassette with the relevant homologous region into the VNP20009 with PKD4
2012.6.13
1. No colony was found on the plate.
2012.6.14
1. Like the last time plenty of small colonies were found on the plate, however none of them was positive.
2012.6.17
1. The kanamycin cassette with the relevant homologous region were successfully amplified by the PCR.
2. Purify the he PCR DNA with bocai kit and elute in 20L dd H2O.
3. Store at -80℃.
2012.6.18
1. Prepare the competent cells for electroporation. (The final concentration of the arabinose was 100M or 10M and the inducible time is 2 hours.)
2012.6.19
1. Introduce the kanamycin cassette with the relevant homologous region into the VNP20009 with PKD4.
2012.6.20-22
1. No colony was found on the plate
2. On the following days, a group of fake colonies were found.
2012.6.25
1. Amplify the the kanamycin cassette with the relevant homologous region with PCR.
2. Purify the he PCR DNA with bocai kit and elute in 30L dd water.
3. Store at -80℃.
2012.6.26
1. Prepare the competent cells for electroporation.(The final concentration of the arabinose was 100M or 10M; the inducible time is 1, 2 , 3 and 4 hours respectively.)
2012.6.27
1. Introduce the kanamycin cassette with the relevant homologous region into the VNP20009 with PKD4 (10-1,10-2,10-3,10-4,100-1,100-2,100-3,100-4).
2012.6.28-29
1. No colony was found on the plate.
2. On the following days a group of colonies were found, however none of them was positive.