1. Twinkle Medicine Hunter
2017/4/11
Twinkle Medicine Hunter
Of Cancer
LZU-China
iGEM Team 2013
Good morning, everyone, we come from Lanzhou University. It is the first time for us to enjoy this fantastic game; and we are glad to be here. Now, we will tell you about a story named twinkle medicine hunter of cancer.
Our presentation includes the following four parts.

2. 1 3 2 4 Content Human Practice Experimental Results Introduction &
Discussion 4
Introduction

3. Background: About Cancer
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Background: About Cancer
Estimated Number of Persons Alive in the U.S. Diagnosed with Cancer in 2006 (N = 11.4 M survivors)
Estimated Number of Female Cancer Survivors in the U.S. in (N = 6.2 M survivors)
Estimated Number of Persons Alive in the U.S. Diagnosed with Cancer on January 1, 2006 by Time From Diagnosis and Gender (Invasive/1st Primary Cases Only, N = 11.4 M survivors
It is well known that, cancer had exceeded cardiovascular disease becoming the top worldwide concern of human being's health.
Cancer cells come from normal cells, what changed? .

4. What cause cancer? Cancer cells come from normal cells, what changed?
Chap Chap Chap Chap.4
Background: About Cancer
What cause cancer?
Cancer cells come from normal cells, what changed?
Gene mutation
There exist oncogenes and tumor suppressor genes. When gene mutated, changes occur in cell cycle, proliferation and apoptosis. Many moleculars and signaling involves, such as, NFKB, JAK-STATs,p53.

5. Activated NFkB were found in almost 70% cancer cells.
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Chap Chap Chap Chap.4
Background: Overview of NF-κB classical signaling pathway
Activated NFkB were found in almost 70% cancer cells.
NFkB signaling pathway has become a variety of
malignant tumor therapy and drug discovery is an
important target.
Activated NFkB were found in almost 70% cancer cells. So, NFkB has become a key signaling pathway that is involed in a variety of malignant tumor therapeutics and drug discovery.

6. NF-κB Signaling Pathway
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Chap Chap Chap Chap.4
Background: Overview of NF-κB classical signaling pathway
FADD
IκB-α
NF-κB
p65/50
cIAP1/2
Bcl-xL/2
Survivin
ICAM-1
Cyclin-D1
VEGF
Gene expression
Cytoplasm
PKC
Nucleu
IKKs
PMA
TNF-α
IL-1
TNFR
IL-1R
MEKKS
TRADD
RIP
TRAFs
TRAF6
TAK1
MyD88
IRAK P Ub
Proteasome
NF-κB Signaling
Pathway
Nuclear Factor κB (NFκB) was discovered by Sen and Blatimore in After 27 years hard working on it, NFKB family has been the hot point in many research fields, such as cancer biology and immunology.
In NFKB classical signaling pathway, in normal cells or the cells are not received external stimulis, IKB binds to p65/p50 heterodimer and shuts down the NFkb signaling, So , the NFkB signaling is inactive.
When TNFa or IL-1 comes to the cell, it subsequently activates IKKs. The acticated IKKs then phosphorylate I-KappaB-Alpha and p65 together with p50, which leads to IkBa ubiquitination and degradation. Latterly, the phosphorylated p65/p50 heterodimer is released and then enters to the nucleus to bind to its binding elements and transcribe target genes.

7. Negative feedback loop
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Chap Chap Chap Chap.4
Background: Negative feedback loop of NF-κB
Negative feedback loop
More importantly, IkB is a target gene of NFkB. So, it shows a negative feedback loop. When p65/p50 activited, expresses IKB, then the expressed IkB will shut-down the signaling, so the expression of IKB up and down, up and down, like water waves.

8. ? Change How to Traditional : the situation LOW accuracy
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Chap Chap Chap Chap.4
Background: Goal
How to
Change
the situation
Traditional :
LOW accuracy
Mice live short/ Can’t take long-term detection ?
Traditional anti-tumor drug evaluation method cannot reflect the effect of drug by measuring every single tumor size. And the common time of therapy of cancer patient lasts 6~12 months while the tumor infected mice model live only 2 months. That is to say, the traditional one can’t make a long term observason. So we gotta change the situation.but how?

9. Pref Suff pSB1C3 Experimental: Device Promoter: 4 copies of κB
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Chap Chap Chap Chap.4
Experimental: Device
Promoter:
4 copies of κB
Binding sequence
IκB+GFP Terminator
Pref
Suff
pSB1C3
In order to improve the sensitivity of evaluation, we amplified the feedback signal through 4 copies of kappaB binding element to express a moderate quantity of IkB GFP protein. Thus, Mice live longer, the characteristics of cancer cell remain, and we could get a long term observation.

10. Why IκBα exists? NF-κB signaling pathway feedback loop dynamis
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Chap Chap Chap Chap.4
Experimental: Design Cancer
Why IκBα exists?
NF-κB signaling pathway feedback loop dynamis
To last a long term detection
Sacrifice less mice
Now you must have some questions about why ikB exists cause as a evaluation systemBecause of the NFkB signaling pathway negative feedback loop dynamic, the tumor issue won’t grow without limit, that's why we add IkB. 

11. Pref Suff pSB1C3 Experimental: Device Promoter: 4 copies of κB
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Chap Chap Chap Chap.4
Experimental: Device
Promoter:
4 copies of κB
Binding sequence
IκB+GFP Terminator
Pref
Suff
pSB1C3
Now, here it comes, IkappaB is the the control part, GFPis the detcct part.

12. NF-κB signal is inhibited.
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Chap Chap Chap Chap.4
Background: Goal
Fluorescent light
Fluorescent light
Time
Time
Oscillation represents drug’s duration time.
NF-κB signal is inhibited.
When accepted mice inoculated, we could pick out IκB-GFP fusion protein every few hours by needle to observe its fluorescence light through smear microscopy. If GFP doesn’t appear, the NF-κB signaling pathway might be inhibited, and there are no active NF-κB binding with its binding element, now we   could confirm that this kind of drug presents inhibitory effect. Besides, the oscillated GFP fluorescence light may reflect the medicine durational time, it's of great significant, we do believe.

13. Results & Discussion: Results Experimental:
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Chap Chap Chap Chap.4
Results & Discussion: Results
Experimental:
DU-145
IκBα
GAPDH
shRNA- IκBα
After constructing the recombinant plasmid ,we choose the nfkb constitutively activated DU145 cells as our work cells. But we must take one factor into consideration. The DU145 cell expresses low level of endogenous expression of IKB. At some times, uncertain stimulations could make the expression of IKB increase sharply. So we have to use shRNA to knockdown endogenous IkB from DU145cells. Western Blot shows that the level of endogenous IkB is totally gone in the knock down cells.

14. Results & Discussion: Results Experimental:
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Chap Chap Chap Chap.4
Results & Discussion: Results
Experimental:
DU-145
IκBα
GAPDH
DU145 Cell
So, we will use this cell lines to transfect IkB/GFP fusion gene and the cells will express Ikb/GFP fusion protein in a moderate level. Western Blot results show that the protein levels in transfected cells are good. And under flurescence microscope, we observe the transfected cells are expressing IkB/GFP fusion protein.
After these work, we can inject the above mentioned cells into node mice to generate mouse model to evaluate candidate drugs.

15. 1. Improve specifity: building the fine-adjusted in other cells.
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Chap Chap Chap Chap.4
Results & Discussion: Future Goals
Experimental:
1. Making our drug screening system work better.
1. Improve specifity: building the fine-adjusted in other cells.
2. Shorten twinkle cycle-time: show some features in pharmacokinetics of drugs.
3. Achieve semiquantitative measure: detect the light intensity of GFP and make a standard curve.
Although the drug screening system we’ve built is representative to a certain degree, we can’t ignore the differences between tumor cells from different tissues and organs. So we could try to build the fine-adjusted system in other cell lines that is useful to improve accuracy during drugs screnning.
The drug screening system which working in a shorter cycle-time may show some features in pharmacokinetics. What we could do is choosing suitable cell lines and optimizing the drug screening system.
We could also detect the light intensity of GFP in actual process of drug screening, and correspond the accurate data of pharmaceutical activity to light intensity. Finally we could achieve semiquantitative measure by building a standard curve about the relationship between pharmaceutical activity and light intensity.

16. 2017/4/11
Chap Chap Chap Chap.4
Results & Discussion: Application
Experimental:
1. How to do: inject DU-145 in which have a moderate expression level of IκB.
2. Long-term drug evaluation system: tumor grow slowly, mice survive longer.
3. Sensitive system: observe twinkle instead o measuring the size of tumor.
1. Making our drug screening system work better.
Although the drug screening system we’ve built is representative to a certain degree, we can’t ignore the differences between tumor cells from different tissues and organs. So we could try to build the fine-adjusted system in other cell lines that is useful to improve accuracy during drugs screnning.
The drug screening system which working in a shorter cycle-time may show some features in pharmacokinetics. What we could do is choosing suitable cell lines and optimizing the drug screening system.
We could also detect the light intensity of GFP in actual process of drug screening, and correspond the accurate data of pharmaceutical activity to light intensity. Finally we could achieve semiquantitative measure by building a standard curve about the relationship between pharmaceutical activity and light intensity.

17. Human Practice Experimental:
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Chap Chap Chap Chap.4
Human Practice
Experimental:
Our team had a interchange with Peking iGEM team and BIT iGEM team in August. We shared our own idea to each other, and offered advice for the problem.
Our Team’s member Li Xiaomeng had been honoured to visit the BIT iGEM 2013 Team’s lab. And help them to characterize and construct a part of Cr sensor.
I’ll introduce our human practice to you. In this summer, we went to Beijing and communicated with BIT and Peking iGEM teams. We interchanged our idea and discussed the problems which we met. After all, it is the first time for us to enjoy the competition. Thus, we learn so much from the exchange.
In August 1st to 15th, we were invited to visit BIT’s iGEM Lab. We helped them to construction and build a part of Chrome sensor.

18. Human Practice Results & Discus Experimental:
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Chap Chap Chap Chap.4
Human Practice
Results & Discus
Experimental:
We held two promotion meetings concerning iGEM for all students in our school and Lanzhou No.1 Meddle School successfully. Meanwhile, the opening day of iGEM lab has been excellently work out with the amazing enthusiasm in this summer vacation.
We also held some events like iGEM opening day and presentations. These had been excellently work out with the amazing enthusiasm in this summer vacation. We believe that a higher number of students would join us and devote themselves to the iGEM competition in the next year.

19. Ph.D Jinbo Yang, Huyuan Feng & Their Labs
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Chap Chap Chap Chap.4
Acknowledgements
Ph.D Jinbo Yang, Huyuan Feng & Their Labs
Institute of Cancer Biology and Drug Screening
of Lanzhou University
School of Life Science of Lanzhou University
Finally, we want to thank our sponsoes ,we made mistakes, we learn much, but they all behind us. Thanks!

20. 2017/4/11
THANKS!
That’s all.Thanks for listening. And welcome to Lanzhou and enjoy the Beef noodle.