Team:ZJU-China/Description

Description

Inspiration from cryptography

The first step

    In order to make a single-letter substitution cipher using E.coli as carrier, the first step is to input information. We have built a light-induced system in E.coli. After being irradiated by corresponding chromatic light, protein kinase which is combined with chromophore will phosphorylate the regulatory protein then, downstream promoter will be activated, realizing the information input process. Information input process is followed by substitution process which needs AND gate, one of the basic logic gate, to complete. We have designed four types of AND gate circuits:
                (1)Red light controlled promoter + lactose promoter +GFP (output)
                (2)Red light controlled promoter + arabinose promoter + RFP (output)
                (3)Green light controlled promoter + lactose promoter + RFP (output)
                (4)Green light controlled promoter + arabinose promoter + GFP (output)

These four AND gate circuits are transferred into E.coli, and they are cultivated together. In the presence of arabinose, the mixed solution could achieve the effect that inputting a monochromatic light will induce bacteria to produce fluorescent protein of the same color. While in the presence of IPTG or lactose, input will induce to produce fluorescent protein in a different color.In practice, for example, when we need to encrypt, we add IPTG, arabinose, IPTG ，arabinose separately into three tubes of mixed solution.

And then irradiate the bacteria using red light, green light, green light, red light.According to the logic gate, the bacteria will emit green light (GFP), green light (GFP), red light (RFP), red light (RFP) which are the encrypted cipher-text.

When we need to decrypt, irradiate them using green light, green light, red light, red light. After the same process, the bacteria will again emit red light (RFP),green light (GFP), green light (GFP), red light (RFP) which means we get the original text.

By now, we have successfully realized the single-letter substitution cipher.

What's more

Although using this method we could realize information encryption easily, its reliability is not high enough. By calculating the inversion frequency, crackers could speculate the encryption rule, and thus decrypt the cipher easily. For the purpose of improving the cipher machine’s safeness, we have been inspired by Enigma, a cipher machine used by the German Army during the Second World War.Enigma was invented by the German engineer Arthur Scherbius, and it’s a common name for a series of electro-mechanical rotor cipher machines. For Enigma cipher machine, the encryption rule will change according to the position of the contents. Similarly, if we are able to change the encryption rule according to time, which is shown in the following figure, then the difficulty of decrypting increase and the cipher machine’s safeness could be guaranteed.

The next step

To describe our system more clearly, we use a double-cycle biological oscillator as a switch working periodically to realize cyclic switching between state 1/2.Oscillation is the repetitive variation, typically in time, of some measure about a central value (often a point of equilibrium) or between two or more different states. In the field of molecular biology, oscillation is a periodic variation of gene expression.
In a complex system or group, it is essential to realize synchronization among all the components and individuals. Coupling within cells could achieve synchronization, help stabilize the target’s state, weaken internal noise and other interference from unreliable components.

To achieve coupling, we select the concentration of two quorum sensing substances, AHL and DPD, as our observed quantity. We achieve periodic fluctuation of concentration through these two molecules’ synthases and degradation enzymes, and their phase difference is controlled at half a cycle. When the concentration of AHL or DPD reaches a certain value, an identification promoter will be activated accordingly. Therefore, state 1 or state 2 can be turned on. When they are adjusted to the appropriate threshold, we can realize cyclic switching between state 1 and 2.

It is hard for oscillation system to maintain stability because of interference from bacteria division, product accumulation and other possible factors. We use micro-fluidic technology to update media in time and constantly, and maintain the quantity of bacteria. We hope we could observe a more stable, and more sturdy oscillation, providing a reliable switch for our cipher machine.

Shown on the left is the actual device we used for our microfludic experiment. The everything you want to know about this chip will be included in Hardware page.

Overall

Part improvement

For better description of single oscillation system, we measured the response intensity of pluxR to AHL.

ZJU-CHINA 2016

ETH_Zurich 2014

We found that the response threshold of pluxR to AHL isn’t exactly as what shown in ETH_Zurich 2014. The minimum response concentration was about 10^-9 M instead of 10^-11M, the maximum response concentration was about 10^-6M instead of 10^-7M. and the relation curve was different with ETH_Zurich 2014 too. Since our samples were much more than ETH_Zurich 2014, we believe that our result are more precise and our model are more in accordance with the reality.

Modeling

Basing on our experiments, we finally establish three model to support our results of experiments. In our light-control model, we research the light input model and the AND Logic Gate Model. The light input shows the relationship between the previous light intensity and the output of the Ccas/R system by varing the production rate of sfGFP, p(t), and the sfGFP abundance, g(t).

And our logic Gate Model use the mathematical equations to simulate the AND relationship between the the concentration of T7 mRNA, I1, and the the concentration of two input substances(In our system, Arbc.), I2.

Our infiltration model shows how important role our microfluidic plays in our experiment. We derive a formula to express the influence of the main channal’s current speed in our oscillation model by analysing the fluid dynamical process and get a ideal result.

Inflating model provides a foundation for our oscillating model, which is the most important basis of our experiments. With the guidance of our pre experiment, we choose the internal AHL concentration to represent the expression of GFP. Basing on the biological reaction process above, we derived the following set of delay-differential equation model for intracellular concentrations of LuxI (I),AiiA (A), internal AHL (Hi), and external AHL (He).

In our models, we finally get a oscillation period about 3h which can be controled by the current speed in microfludics. In our light-dependent experiment, the optical response time is nearly 3h, which supplies the proof of the interconnection between the oscillation and light-dependent control.

Reference

Light induced system：
1）Tabor J J, Levskaya A, Voigt C A. Multichromatic Control of Gene Expression in Escherichia coli[J]. Journal of Molecular Biology, 2011, 405(2):315-24.
2）Levskaya A, Chevalier A A, Tabor J J, et al. Engineering Escherichia coli to see light[J]. Nature, 2005, 438(7067):441-2.
3）Olson E J, Hartsough L A, Landry B P, et al. Characterizing bacterial gene circuit dynamics with optically programmed gene expression signals.[J]. Nature Methods, 2014, 11(4):449-455.

Logic gate：
Anderson J C, Voigt C A, Arkin A P. Environmental signal integration by a modular AND gate[J]. Molecular Systems Biology, 2007, 3(1):: 133.

Oscillation：
1）Prindle A, Samayoa P, Razinkov I, et al. A sensing array of radically coupled genetic/biopixels/'[J]. Nature, 2012, 481(7379): 39-44.
2）Danino T, Mondragón-Palomino O, Tsimring L, et al. A synchronized quorum of genetic clocks[J]. Nature, 2010, 463(7279): 326-330.

Contact Us
Room 413,Biology lab center, Zijingang Campus
Zhejiang University, YuHangTang Road NO.866
Hangzhou, China

iGEM ZJU-China 2016 Team
igem_zjuchina_2016@outlook.com