Overview

     The design of our engineered bacteria contains four major modules: Production Module, Oscillator Module, Directed Evolution Module and Safety Module. We plan to choose a probiotic with natural anti-depression capacity and then construct the above modules in it. With such four modules integrated together, our probiotic is purposed to work synergistically to ease depression.

Choice of probiotic strain

    Some particular strains of probiotic Bifidobacterium longum (B. longum), like NCC3001, have been shown to have the capacity to reduce depression scores and alter brain activity [1]. Meanwhile, they can act as a common probiotic beneficial for patients. There have been many commercial B. longum probiotic products on sale (Figure 2). So we planned to use one of the B. longum strains among these as our engineered bacteria inhabited in intestine to work. However, at the development stage of the project, we plan to use Bacillus subtilis 168, which is also a Gram-positive bacteria as B. longum, as the model to verify the functions of the modules. Since Escherichia coli is easier to manipulate, we also tested some modules in E. coli before moving to B. subtilis.

Production Module

    S-adenosyl-L-methionine (SAMe) is the antidepressant molecule we chose to reduce depression symptoms and what this module aims to produce. We found an SAMe synthetase Sam2, which doesn’t have a feedback inhibition phenomenon, from Saccharomyces cerevisiae (S. cerevisiae) to synthesize SAMe [3]. As for the secretion of SAMe out of bacteria, we found a passive transporter Pet8p in S. cerevisiae mitochondria.
    Since our engineered bacteria are gram-positive bacteria, we choose Bacillus subtilis 168 (B. subtilis 168) as our model bacteria to verify our parts as said above. As Sam2 is purposed to express in gram-positive bacteria, we firstly optimized its sequence to gain a new part opSam2 (BBa_K4144011). To express this SAMe synthetase opSam2, we used IPTG-induced Pgrac as the promoter of 6xHis::opSam2 (Fig. 3A), and tested its expression and function in Escherichia coli DH5α (Ε. coli DH5a) and B. subtilis 168. The His-tag can be used to verify protein expression in immunoblots. As for the transporter Pet8p, we also optimized it to gained a new part opPet8p (BBa_K4144004), and then used Xylose-induced promoter Pxyl to regulate its expression.

    Meanwhile, to ensure that opPet8p can integrate into bacterial membrane, we fused it with a B. subtilis signal peptide Mistic which has also been used to facilitate membrane protein expression in E. coli [4], and we optimized it to opMistic as well. To visualize the localization of opPet8p, we used sfGFP as another tag at the C-terminus. With the N-terminal 6xHis tag, we constructed the expression vector for the fusion protein 6xHis::opMistic::opPet8p::sfGFP (Fig. 3B), and tested its expression and membrane localization in Ε. coli DH5a and B. subtilis 168. After verifying separately, these two components will be co-transferred and tested for the secretion of SAMe out of the bacteria as the proof-of-concept for the Production Module. For the final probiotic bacteria, the transporter opPet8p will be constitutively expressed, and the synthetase opSam2 will be periodically expressed under the control of the oscillator.

Oscillator Module

    We chose the REPRESSILATOR (BBa_K3482025)[5] as the oscillator to achieve periodic SAMe secretion. This oscillating system contains three proteins, namely, λCl, TetR and LacI. These three proteins are all transcriptional repressors that can inhibit the expression from their corresponding promoters, and these three proteins are expressed under the drive of these three promoters. This enables the three protein concentrations to change periodically respectively.

    For the experimental design for this part, we plan to conduct experiments in B. subtilis to verify the functions of these three proteins and observe whether these three proteins can still function as in E. coli and inhibit their corresponding promoters. In addition, we also intend to reconstruct the REPRESSILATOR directly in E. coli and B. subtilis, to observe whether it can generate oscillation. For the final probiotic bacteria, the SAMe synthetase opSam2 will be controlled by pLacI. For the proof-of-concept experiments, the reporter GFP is inserted downstream of pLacI instead to monitor the oscillation of the system.

Directed Evolution Module

    We planned to develop a LacI protein that could tolerate high-level lactose without losing repression on the promoter. For this purpose, we designed the Directed Evolution Module. The basic principle of directed evolution is shown below: building rounds of mutagenesis, selection and amplification, that is, generating a library of variants, separating target desired members by setting multiple conditions and then enlarging them as the templates of the next round.

    In order to select desired LacI, we constructed an expression element where two selection proteins are placed downstream of LacI promoter. The first one is SacB protein (BBa_K22921). This part encodes the Bacillus subtilis levansucrase which catalyzes the hydrolysis of sucrose and synthesis of levans, which is lethal to gram-negative bacteria such as E. coli. The second one is KanR, which provides the resistance to kanamycin. We also included a reporter mRFP1 by using BBa_J04450 as vector backbone of our plasmid, of which fluorescence could aid in the judgement. In addition to these, a LacI controlled by a constructive promotor (BBa_J23116) is also included. The schematic diagram is showed below (Fig.6).

    We first used saturation mutagenesis to build the variant library of LacI protein. By overlap extension PCR, we plan to introduce mutagenesis into three different residues, I79, F161 and L296, which are located in the binding pocket of IPTG [6]. Then we combined three fragments together, using it as a megaprimer to amplify our constructed plasmid [7]. By transforming these plasmids with mutant LacI into E. coli, the protein selection is carried out by a selective culture of the transformed bacteria. We are now able to select different LacI proteins by simply shifting the culture condition of the E. coli.

    In line with the plasmid design, we set up different culture conditions to select the desired LacI mutants. By adding sucrose and gradually lifting the level of lactose into the culture plate, we eliminate variants that couldn’t resist a certain level of lactose, isolating the members that contain desired LacI mutants. At the same time, some variants that may lose the ability to bind lactose and release from promoter permanently would also be sifted out by other selection conditions containing kanamycin and lactose. Among the “survivals”, a new round of amplification and selection will be carried out, until the lactose level reaches the desired level. These “survivals” will be sequenced for the LacI mutants they possess. Finally, measurements will be carried out to verify the sensitivity of these mutants.

Safety Switch Module

    Considering we are designing engineered bacteria that will be used in human gut, the safety of our engineered bacteria should be stressed. On the one hand, our patients should be able to remove the bacteria actively as their wish. On the other hand, the bacteria should be prevented from leaking into environment. Thus, we’ve designed a parallel safety switch to solve these problems (Fig. 7) [8, 9, 10].

    First, we used a chemical switch (BBa_K4144081) for the active termination. CinR is an autoinducer-binding protein found in Rhizobium leguminosarumis. In complex with the quorum sensing molecule O3-C14 HSL, which is synthesized by the inducer CinI, CinR activates the pCin promoter. Holin is a protein that will make holes on cell membrane and cause the death of bacteria. We use pCinR as a promoter for holin so that the molecule O3-C14 HSL can activate suicide by inducing the expression of holin.
    Next, we use a temperature switch (BBa_K4144082) for leakage protection. TlpA36 is a temperature sensitive protein which is capable of repressing the pTlpA promoter under temperature below 36 degree Celsius, thus repressing the expression of PenI repressor. This will activate the pPenI promoter, which controls the expression of holin. As a result, holin will express below 36 degree Celsius and cause the death of bacteria.
    Before combining these two promoters, we made constructs to test them separately both in E. coli and B. subtilis.

Reference

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