Back to top

Proof of concept

Overview


    Through a year’s preparation and lab work, we’ve achieved basic proof of concept via several fundamental experiments, hardware and models.
     To develop a novel depression therapy, we planned to engineered probiotic Bifidobacterium longum (B. longum) with natural anti-depression capacity to generate natural antidepressant S-adenosyl methionine (SAMe). We choosed Bacillus subtilis (B. subtilis) as the experimental gram-positive bacterial model to engineer. We have engineered such strain which could synthesize SAMe in high efficiency (BBa_K4144008 and BBa_K4144011), and had capacity to secrete SAMe (BBa_K4144004, BBa_K4144006 and BBa_K4144007). The production of SAMe can be regulated by oscillator (BBa_K3482025) module, which will ensure that drug plasma concentration remains for a period of time and reduce side effects. To make our oscillator not disrupted by lactose in human intestine, we designed a directed evolution pipeline to improve the sensitivity of LacI (BBa_K4144041) to lactose. Meanwhile, we added parallel safety switch to ensure the biosafety (BBa_K4144081 and BBa_K4144082).
     We will demonstrate that our designs are able to play a role in the following content.

SAMe Production

    In this part, to generate anti-depressant S-adenosyl methionine (SAMe), we successfully expressed exogenous optimized SMAe synthetase opSam2 in Escherichia coli (E. coli) and B. subtilis (Fig.1). Using the BCA quantification (Fig. 1A, B), we make the brightness of land comparable. There is a positive correlation between expression level of opSam2 and induction time in E. coli (Fig. 1C) and in B. subtilis (Fig. 1D).

Figure. 1 Expression verification of opSam2 in E. coli and B. subtilis through WB.


    Then, to characterize the enzymatic activity of opSam2, we used enzyme-linked immunosorbent assay (ELISA) to measure the concentration change of SAMe in Experiment Group (pHT-opSam2) and Control Group (Empty Vector) after induction. As the figure. 2 shows, there is a positive correlation between the induction time and the increase of relative concentration of SAMe.
    Combined with the induction expression of opSam2, we can conclude that the expression of opSam2 can facilitate the generation of SAMe, and the basic part BBa_K4144011 and composite part BBa_K4144008 works in B. subtilis. It would be expected to work in B. longum as well.

Figure. 2 Enzymatic activity verification of opSam2 in B. subtilis.


SAMe Secretion

     In this part, to secret our anti-depressant SAMe from cytosol to extracellular environment, we successfully expressed exogenous SAMe transporter opPet8p from mitochondron of Saccharomyces cerevisia, in E. coli and B. subtilis (Fig. 3). We used both 6xHis as a N-terminus tag and sfGFP as a visual tag and C-terminus tag. Using two kinds of antibodies respectively (Fig. 3D), we can verify the successful expression of opPet8p (Fig. 3B and C).

Figure. 3 Expression verification of opPet8p through WB against N-terminus and C-terminus


    To secrete SAMe, SAMe transporter opPet8p is required to localize in bacterial membrane, as in the mitochondrial membrane in yeast. Thus we performed fluorescent microscopy to verify its membrane localization. We reduced the induction time and concentration of inducer xylose to weaken the fluorescent signal, so that we can find its membrane localization (Fig. 4). And we suppose that opPet8p might have membrane localization with such signal distribution and it would be expected to have transportation ability as well.

Figure. 4 Membrane localization of opPet8p::sfGFP.

Oscillator Regulation

    In this part, first of all, we tested whether an oscillation cycle can be obtained in E. coli by co-transformation of the Repressilator plasmid and a reporter plasmid (which consists of an GFP driven by Lactose promoter). We showed that there is indeed fluorescence emitted by GFP in the doubly-transformed E. coli, and with the aid from math modeling group, we had determined that length of the oscillation cycle is about 60 mins (Fig. 5).

Figure. 5 Oscillator cycle determination in E. coli


    Another experiment that we would like to try is to transform the Repressilator plasmid and the reporter plasmid mentioned above into B. subtilis, and see if an oscillating fluorescence signal can be observed. But due to the lack of time, we haven’t got our hands on this project. But nevertheless, we do believe results strongly in favor of our hypothesis will result if this experiment was carried out, due to the fact that the three transcription factors and there corresponding promoters are thoroughly studied, and the fact that there has been publication confirming the success of protein expression in the chassis.

Directed Evolution

    In this part, we have successfully constructed the selection plasmid containing three reporter genes (Fig. 6A) and further built up the LacI site-mutagenesis library using MWGAWHOP (The Megaprimer PCR of whole plasmid), suggested by the obvious contrast between two groups after DpnI digestion (Fig 6 B-C).

Figure 6 Plasmid construction and mutagenesis.
A: Colony PCR shows the existence of three genes on plasmid.
B-C, Mutagenesis library construction.
A: No megaprimer in MEGAWHOP.
B: Megaprimer added in MEGAWHOP. DpnI digestion is applied to both group, and non-mutated plasmid can’t be transformed into competent cells.

    After round of selection, we successfully identified the ideal mutant Strain #29 (Fig.7C) that only responds to high-level of inducer, with almost no leakage itself. Sequencing of the mutant also suggests that other highly possible residues contribute to the substance binding of LacI repressor.

Figure 7 Selection results.
A: Control group 1. Original strain and random mutant are applied on empty antibiotic plate with IPTG contrast.
B: Control group 2. Original strain is applied on Kan+ plate with IPTG gradient.
C: Desired mutant. No colony growth is observed at 0 and 0.1mM IPTG, suggesting that the LacI can tolerate high-level IPTG.

Safety Switch

    In this part, we constructed two composite plasmids respectively as chemical-induced switch (BBa_K4144081) and temperature-controlled switch (BBa_K4144082) to ensure biosafety. In addition, plasmid pET-CinI has been constructed to produce an inducer to start chemical killing switch. As we expected, low temperature could activate thermo-switch, while for the chemical-induced switch there was no signal in E. coli after being induced by CinI,

Fig 8. There was green fluorescence when BBa_K4144082 was induced by low temperature


    In addition, to verify whether PBSX holin (BBa_K1054002) can kill the host bacteria, we qualitatively and quantitatively tested the function of this bacteria toxin expressed in E. coli. The results indicated that the expression of holin could lyse bacteria to some extent under the conditions we needed.

Figure 9. Inducing the expression of BBa_K1054002 in E. coli BL21.
A. Induction on the culture plate
B. Change in turbidity

Hardware

    We have completed the design of the portable dual port filter. Through 3D printing, finally, we can get the solution containing the intensified Escherichia coli, which shows the effectiveness of the product.
    In addition, we have realized a simple enteral drug simulation device, which successfully realizes the peristalsis of the intestine and the flow of fluid. The effect is as follows.

Model

    We built the entire system with SimBiology, simulating our design concept. Moreover, we modeled the oscillator module and our second model—Molecular concentration prediction model in detail by using differential equations and provided the experimental group with suggestions on how to improve the stability and period of the oscillator as well as how to improve the secretion rate and satisfy the minimum concentration limits.