The core targets of our project will be implemented by engineered Escherichia coli. It consists of three modules, and the main module "Colonization & Blocking" can achieve the effect of alleviate allergy symptoms caused by cats. Additionally, we design "Sterilization" module to ensure the health of cats in our project. At the same time, in order to ensure the safety of the project, multiple sets of "Suicide" modules have designed for our engineered bacteria.

The "Colonization" module can help engineered bacteria survive in the cat mouth for a long time by expressing adhesion protein Hsa on the surface of bacteria, which is conducive to the subsequent "Blocking" module work.

The "Blocking" module is the real core of the project. This module includes two single-chain fragment variables (scFvs), NeuA and NeuB, which function together to bind the allergen Fel d 1 and prevent IgE engagement. So people who are allergic to cats can interact their cats without developing allergies.

In order to avoid the biofilm formed by engineered bacteria using by oral pathogenic bacteria Streptococcus mutans, the "Sterilization" module can detect the concentration of S. mutans and then express the protein ClyR which can kill S. mutans to maintain oral health of cats.

The "Suicide" module mainly adopts the strategy of realizing suicide by sensing the difference between the temperature of the oral and the environment. Additionally, it also senses the difference between the pH of oral and digestive tract to realize suicide.

We design the "Colonization & Blocking" module as an NOT-Gate. We utilize the mechanism of quorum sensing, and when the engineered bacteria are in short supply, they express adhesion proteins that are better able to persist in the oral environment. When the number of engineered bacteria are sufficient, The TetR protein is expressed and it binds to TetA promoter to inhibit the expression of adhesion protein, and at the same time engineered bacteria begin to express scFvs.

Colonization

According to reports in the literature, Streptococcus gordonii str. Challis is able to colonize the tooth surface of healthy humans, mainly due to the secreted Hsa protein can specifically bind to sialic acid-containing glycans. Hsa is Siglec-like binding regions (SLBRs) which can adhere to protein-attached carbohydrates. Additional forms of sialic acid and alternative linkages are found in animal sialoglycans [1], it means that engineered bacteria can utilize Hsa to colonize the cat mouth.

We genetically engineer E. coli to create bacteria extracellular matrix material by genetically appending Hsa domains to the amyloid protein CsgA, the dominant proteinaceous component in E. coli biofilms [2,3]. Engineered bacteria express CsgA-Hsa to the surface of the cytomembrane so that it binds firmly to the surface of cat tooth.

Blocking

In previous literature reports, researchers utilized recombinant cat allergen recombinant dimeric Fel d 1 to immunize VelocImmune mice to produce corresponding polyclonal antibodies, and verified the two monoclonal antibodies with the best blocking effect [4].

Based on this literature, we obtained the light chain and heavy chain genes of these two monoclonal antibodies, fused the light chain and heavy chain through rigid linker to obtain scFvs NeuA and NeuB, and fused OmpA signaling peptides at the N-terminal of NeuA and NeuB to helps secrete NeuA and NeuB into periplasmic space. Engineered bacteria can express NeuA and NeuB to bind the feline allergen Fel d 1 in the mouth of cats, so that Fel d 1 is no longer able to bind to IgE in people who are allergic to cats and cause allergy.

It is worth mentioning that the removal of cat allergens in the mouth has not been found to have any harm to cats, so we have confirmed the feasibility of our project [5,6].

NOT-Gate

Vibrio cholerae uses a quorum-sensing (QS) system composed of the autoinducer 3,5-dimethylpyrazin- 2-ol (DPO) and receptor VqmA, which together bind to Promoter qtip and activate transcription [7]. According to reports in the literature, we found that E.coli itself can produce DPO [8], and the concentration of DPO is positively correlated with the amount of E. coli.

When the number of engineered bacteria is insufficient, bacteria will express Hsa-CsgA, which is in the downstream of tetA promoter. With the growth of bacteria, DPO accumulates. Then VqmA will combine to qtip promoter and activate transcription of TetR, NeuA and NeuB. TetR will represses the transcription of Hsa-CsgA.

In this way, engineered bacteria express a large number of Hsa-CsgA proteins in the early stage of growth so that they can survive in the mouth for a long time. When the concentration of engineered bacteria reaches a certain level, scFvs will begin to be express to neutralize of Fel d 1. And the expression of Hsa-CsgA would be shut down, in order to prevent damage to feline oral health. When the concentration of engineered bacteria decreases again, Hsa-CsgA will be expressed again.

To prevent the biofilm produced by engineered bacteria is used by some pathogenic bacteria in the cat's mouth [9], we design “Sterilization” module. This module works by sensing the concentration of S. mutans and expressing the protein ClyR which can kill S. mutans.

CSP is a quorum sensing signaling molecule of S. mutans, and its concentration is positively correlated with the amount of S. mutans. ComD is a membrane-bound histidine kinase (HK) capable of autophosphorylation in response to sensing CSP. The response regulatory factor (RR) ComE then catalyzes the transfer of phosphorylated groups to aspartic residues in its receptor region. Phosphorylated ComE (P-ComE) is allosterically transformed into an active conformation that binds upstream to the CSP-sensitive promoter nlmC and activates transcription [10]. Then expressing ClyR. ClyR, a chimeric lysin, is composed of a catalytic domain from PlyC and a cell-wall binding domain from PlySs2. ClyR can recognize and lyse the cell wall of S. mutans and avoids the biofilm formed by S. mutans on the surface of our bacteria [11] .

To avoid the potential contamination caused by the leakage of engineered bacteria into the environment, we put forward several optional schemes of the “suicide” module.

Considering oral temperature of cats is normally higher than RT, we decided to enable the bacteria to kill themselves at low temperatures and only survive at oral temperature. To achieve such a function, we utilized RNA thermometers and the RelE toxin to build one module [12,13].

Additionally, Considering oral pH of cats is different to cats' digestive tract, we decided to enable engineered bacteria to kill themselves at low pH. Engineered bacteria will continuously express HepT toxin and MntA antitoxin which neutralize each other without causing toxicity [14]. When our bacteria enter the digestive tract, due to the decrease in pH, acid-Inducible promoter asr will activate transcription [15]. Then expressing lambda repressor to bind promoter R which inhibits MntA transcription. HepT can perform suicidal function to avoid the leakage of engineered bacteria through digestive tract.

Although the ultimate goal of our project is benefit to people who are allergic to cats, we still consider the health of cats. Streptococcus salivary K12 is an oral probiotic which can inhibit halitosis and caries pathogens to a certain extent [16,17], so we want to further use Streptococcus salivary K12 as our chassis organism.

Additionally, due to time and experimental constraints, our project did not actually conduct in vivo. But our instructor Mingqian Feng advised that we can conduct preliminary experiments in rats firstly in our future work. Further experiments will be carried out after the desired results are obtained.

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