Team:UCAS-China/Proof Of Concept

• Home
• Project
  • Description
  • Design
  • Proof Of Concept
  • Demonstrate
  • Future Plan
  • Engineering
  • Contribution
  • Parts
• Hardware
• Model
  • Model for the Design
  • Dynamic of Hp Infection
  • Dynamic of Hp-SHEEP-Human System
  • Model for Therapy Method
  • Estimation for Individuality in Stable States
• Human Practices
  • Integrated Human Practice
  • Education&Public Engagement
  • Collaborations
  • Implementation
  • Partnership
• Team
  • Meet The Team!
  • Attributions
  • Notebook
  • Acknowledgement

<div> </div> <div> </div> <div> </div> <div> </div>

Proof Of Concept

LL-37 vs. H. pylori

It is vital that LL-37 should be provided without redundancy or lack. Therefore, we have to make our drug application targeted and moderate. So, we have to apply LL-37 in situ, i.e. in stomach. Considering that LL-37 produced by human gastric epithelial cells significantly inhibits the reproduction of H. pylori, we came up with an idea of providing LL-37 by introducing engineered bacteria that produce the cathelicidin regularly and safely into gastric flora in order to imitate the natural flora regulating process.

NOT+NOT=AND

NtrC is used as the protein in our chassis. The mechanism of its regulatory function has been well studied.

the figure is from [5]

The figure above shows NtrC carries out its biological function by binding with the helix of DNA at a precise position, further causing the helix bending between enhancer and promoter (glnAp2) and then enhancing transcription.

the figure is from UCAS team in 2017

This figure shows the NtrC regulating mechanism on a larger scale. When the ammonium concentration is low, NtrC is constantly activated by NtrB and binds to DNA helix to start transcription. However, when the ammonium concentration goes up, NtrC will be inactivated, therefore it can no longer bind to DNA and the promoter glnAp2 will be inhibited. Considering their adequate experimental evidence, the NtrC-glnAp2 part will work.

Hence tetR/tetA promoters are widely used as promoters inhibited by the expression of TetR gene, we believe they are a competent NOT gate switch when working together with TetR.

We preliminary decide to use Stenotrophomonas maltophilia Endolysin P28 as the lysis protein. In fact, endolysin is naturally used by phages to lyse host cells. Through a big body of studies, people have almost fully understood how endolysin is expressed and how it lyses the cell membrane. However, many kinds of endolysin just lyses Gram-negative bacteria. Since our engineered bacteria L. acidophilus is Gram-positive, it is necessary for us to choose an endolysin that works in Gram-positive. It is reported that Stenotrophomonas maltophilia Endolysin P28 shows antibacterial activity against both Gram-negative and Gram-positive bacteria.

Given these evidences we believe that once the upstream genetic parts work, the lysis part will work smoothly and meet our expectations.

About Biosafety

In order to protect natural ecosystems also our patients for microbial therapies, and address public concern, biosafety is a critical concern in the development of our Sheep. We concerned biocontainment, avoiding unintended proliferation into the environment, also biosafety, which we mainly concerned avoiding its escaping from the specific lesion to other healthy parts of the body. Current strategies mainly include integrating toxin/antitoxin ‘kill switches’ and establishing auxotrophies for essential compounds. However, either of them has its own obstacles. This year, we provided our own solutions and improvements to address the risks that may cause the leakage of our gene. By combining our high-performance cold-inducible on-switch with the toxin system, we optimized the ‘kill switch’ from its foundation, improving the response speed also the efficiency, so that our sheep will have a strong control of the baobab tree.

The function of cold-inducible ON-switches

Within these screening circuits, the expression of GOI (goal of interest) was inhibited by the evolved transcription factor and the evolved protease could activate the reporter gene by cleaving the sensitive repressor.

The cold-inducible ON-switch is encoded on two plasmids. The circuit is shown with the genetic parts and relationships among them.

We got the temperature-sensitive parts TEVts and CI434ts-TEVsite from our advisor. TEVts is TEV protease from Tobacco Etch Virus which can recognize special amino acids sequence (ENLYFQ) and cleave the sequence at the site between F and Q. Its activity is triggered by coldness.

CI434ts-TEVsite is a thermo-sensitive transcription repressor. We insert the TEV recognition sequence into the link part of transcription factor so that TEV protease can cut the transcription factor to make it inactive.

we put the TEV protease coding gene under the promoter of corresponding CI434 so that TEV and CI434 inhibit each other. Once there is a small disturbance, the balance will lean to one side to act. We put our target gene under the promoter of pCI434, so that its expression is controlled by the transcriptional factor side. When coldness comes, the TEV protease is active and cut the transcription factor to relieve the inhibition of promoter so that the target gene can be expressed.

The cold-inducible TEV protease mutants and heat-inducible transcription factors mutants are all evolved through a suitable restriction, which creates many different switches with a wide range of transition temperature.

We chose two kinds of cold-inducible ON-switches, consisting of TEVts#6 and TEVts#18 correspondingly, to measure their temperature response curve more precisely by flow cytometer in TOP10 strain. From Figure 4 we can see that two group both show narrow transition ranges and have ∼100-fold induction, which was achieved within less than ten degrees. Thus, our cold-inducible ON-switches show high performance and versatility, which ensures the potential for basic research, as well as industrial and biomedical applications, and truly makes engineered bacteria precisely controlled.

The induction curve of the cold-inducible ON-switches (TOP10)

In our design, we plan to colonize engineering bacteria into the stomach through capsules. After the engineering bacteria identified the position of H. pylori through the ammonia concentration induction pathway, the anti-bacterial peptide was released by self cleavage to inhibit the growth of H.pylori. When H. pylori leaves the human body, the biosafety switch can recognize the decrease of environmental temperature and cause cell suicide with toxic protein. However, in practice, promoter has a certain chance to mistakenly turn on the transcription of toxin protein under the condition of constant ambient temperature, and secrete toxin protein, which cause cell suicide. Such leaky expression will lead to the decomposition of engineering bacteria and reduce the actual concentration of engineering bacteria, and then affect our treatment efficiency.

The control system of leaky expression

SsrA is a kind of degradation tag in organism, which can make proteins be degraded after the process of a wrong translation by adding it to the C-terminal of protein. We connect a SsrA degradation tag to DOC gene through the TEV protease cutting site. If Doc-gene is occasionally expressed under the condition of constant environmental temperature, due to the existence of SsrA, the enzyme system of bacteria can recognize and degrade the toxin protein, so as to prevent it from harming the cells. When the temperature is reduced, TEV protease is activated, and the TEV site connecting Doc gene and SsrA degradation label is cut, so that the translated toxin can kill cells and prevent biocontainment.

Reference

[1] M.R. Atkinson, N. Pattaramanon, A.J. Ninfa. Governor of the glnAp2 promoter of Escherichia coli. Mol. Microbiol., 46 (2002), pp. 1247-1257.

[2] Hervas AB, Canosa I, Little R, Dixon R, Santero E. NtrC‑dependent regulatory network for nitrogen assimilation in Pseudomonas putida. J Bacteriol. 2009;191:6123–35