Overview

The wet experiment was designed to demonstrate the engineering feasibility of our blue light-controlled expression system to synthesize the odour substance phenylethanol (2-PE), which can modulate sadness. In this section we present the results of experiments in four aspects: the blue light-responding switch, the blue light-controlled system, the red light-responding switch, and the suicide system. The success of the blue light-responding switch showed that we could control the expression of downstream genes through a blue light switch, and the validation of the blue light-controlled system illustrated that our project had the ability to synthesize 2-PE. Together with the successful validation of the red light-responding switch, the suicide system could be constructed under the control of red and blue light, lying in the foundation for further expansion of other light sources corresponding to the regulation of more emotions.

1. Blue Light-Responding Switch

In the blue light-responding switch, we constructed the pSB1C3-stuffer-pro plasmid (BBa_K4427009), which includes the blue light sensitive protein EL222 gene and the red fluorescent protein gene (mRFP1). It expresses EL222 protein with the induction of L-arabinose, and under blue light, EL222 dimerize to bind to the P_Blind promoter, initiating the expression of the downstream mRFP1. By detecting the presence of mRFP1, we could verify that the blue light-responding switch was working properly.

1.1 Agarose Gel Electrophoresis and Double Digestion

To ensure that the EL222 protein gene (BBa_K4427003) and mRFP1 (BBa_K4427006) gene were inserted in the correct position, and to verify that the plasmids were correctly transformed, we performed plasmid extraction, double digestion (XbaI and SpeI), and agarose gel electrophoresis on DH5α bacteria solution after transformed with the pSB1C3-stuffer-pro (BBa_K4427009). The results of the electrophoresis after plasmid extraction showed that our plasmid was successfully transformed into BL21, and the results of the XbaI and SpeI double digestion experiments showed that our target gene was inserted into the target site correctly.

1.2 The Blue Light-Responding Switch Control the Expression of the Red Fluorescent Protein mRFP1

After demonsrating that the plasmid was correctly constructed and successfully transformed, we verified the light-sensitive function of the pSB1C3-stuffer-pro plasmid (BBa_K4427009) in a dark room. With reference to the literature, we performed periodic experiments on engineered bacteria BL21 in a dark room with blue light irradiation for 2 hours and dark treatment for 2 hours alternatively, for a total of 8 hours, and a control experiment was conducted in complete darkness. The experimental results showed that the engineered bacteria containing the pSB1C3-stuffer-pro plasmid (BBa_K4427009) successfully expressed mRFP1, indicating the efficacy of our blue light-responding switch.

1.2.1 SDS-PAGE of mRFP1

We performed SDS-PAGE on both sets of bacteria and obtained the following result. mRFP1 has a molecular weight of about 25 kDa, which is comparable to the molecular weight of the marker corresponding to the band in the electrophoresis graph (Fig. 3), tentatively proving the successful expression of the target protein in the engineered bacteria.

1.2.2 The expression levels of red fluorescent protein

We used a UV lamp to irradiate the two groups of bacteria solution and the results showed (Fig. 4) that a clear fluorescence appeared in the experimental group while no fluorescence appeared in the control group. We observed the microstructure of the bacterial solution for the experimental group under a laser confocal microscope (Fig. 5). These results showed the engineered bacteria synthesized red fluorescent protein under the control of the blue light-responding switch.

1.2.3 Determination of Fluorescent Red Fluorescent Protein mRFP1 by Fluorescence Spectrometry

In order to determine whether the protein expressed by the engineered bacteria was the mRFP1 we expected, we performed fluorescence spectroscopy on the bacteria solution. The results (Fig. 6) showed the protein had an excitation wavelength of 584 nm and an emission wavelength of 612 nm, consistent with the standard mRFP1. It determined that our engineered bacteria successfully synthesized mRFP1 under the control of the blue light-responding switch.

In summary, we successfully verified the validity of the blue light-responding switch. Also, we found the control group (Fig. 7) also had a lower emission peak. Such a leakage expression phenomenon is of concern. But for time reasons, we did not explore the leakage expression further.

2. The Red Light-Responding Switch

In the red light-responding switch, we constructed pET-22b-RLP ( BBa_K4427016), which includes Ho1 (BBa_K4427010), the red light sensitive protein BphP1 (BBa_K4427000) and the deterrent protein PpsR2 (BBa_K4427001). It expresses Ho1, BphP1 and PpsR2 under the induction of IPTG. Ho1 catalyzes heme to BV, BV binds to BphP1 and acquires light-sensitive ability, and can bind to PpsR2 under 760nm NIR light irradiation to inhibit the suppression of PpsR2, highly expresses downstream genes. While under 680nm red light irradiation, the suppression effect is quickly disabled and suppressed the expression of the downstream gene again. To validate the above function of our red light initiation system pET-22b-RLP (BBa_K4427016), we performed the following validation:

2.1 Agarose Gel Electrophoresis and Double Digestion

To ensure the BphP1 and PpsR2 genes (BBa_K4427018) and the mutant green fluorescent protein (sfGFP) gene (BBa_K4427011) were inserted in the correct positions, we performed plasmid extraction, double digestion (XhoI and NdeI), and agarose gel electrophoresis on DH5α bacteria solution after transformed with the pET-22b-RLP plasmid(BBa_K4427016). The results showed (Fig. 9) that our target gene was transformed successfully and inserted into the target site correctly .

2.2 The Red Light-Responding Switch Control the Expression of Green Fluorescent Protein Mutant sfGFP

After demonsrating that the plasmid was correctly constructed and successfully transformed, we verified the light-sensitive function of pET-22b-RLP (BBa_K4427016) in a dark room. With reference to the literature, we performed irradiation experiments experiments on engineered bacteria BL21 with far-red light irradiation for 4 hours and a control experiment with complete light-proofing. After the irradiation, the following observations were made on the bacterial solution.

2.2.1 The expression levels of green fluorescent protein

We used a UV lamp to illuminate the two groups of bacterial fluids and the results (Fig. 10) showed that clear fluorescence appeared in the experimental group, while no fluorescence appeared in the control group.

2.2.2 Fluorescence spectra of sfGFP

To determine whether the protein expressed by the engineered bacteria was the green fluorescent protein (sfGFP) that we expected, we performed fluorescence spectroscopy on the bacterial solution. The results (Fig.13) showed the protein had an excitation wavelength of 488 nm and an emission wavelength of 510 nm, which is consistent with the standard sfGFP. It determined that our engineered bacteria synthesized sfGFP successfully. However, the results (Fig.12) of the fluorescence spectra of the bacterial broth in the light-avoidance group also showed that sfGFP was synthesized and that leakage of gene expression may have occurred.

For our project, the red light repression function of the BphP1-PpsR2 system was not indispensable. Due to time constraints, we only performed experimental validation of the activated expression function and did not complete functional validation of the repressed expression function.

3. The Blue Light-Controlled System

In the blue light-controlled system, we constructed the pSB1C3-stuffer-Lp plasmid (BBa_K4427002). It expresses EL222 protein with the induction of L-arabinose, and under blue light, EL222 dimerize to bind to the P_Blind promoter, initiating the expression of the downstream ethanol dehydrogenase (Adh1) and alpha-keto acid decarboxylase ( KdcA),and catalyzes the synthesis of the substrate phenylpyruvate into phenylethanol (2-PE). To validate the above functions of our blue light expression system pSB1C3-stuffer-Lp plasmid (BBa_K4427002), we performed the following validation.

3.1 Agarose Gel Electrophoresis and Double Digestion

To ensure that the EL222 protein gene (BBa_K4427003), the Adh1 gene ( BBa_K4427004) and the KdcA gene (KdcA BBa_K4427005) were inserted in the correct positions and the plasmids were correctly transformed, we performed plasmid extraction, double digestion (XbaI and SpeI), and agarose gel electrophoresis on DH5α bacteria solution after transformed with the pSB1C3-stuffer-Lp(BBa_K4427002). The results of the electrophoresis after plasmid extraction showed that our plasmid was successfully transformed into BL21, and the results of the XbaI and SpeI double digestion experiments showed that our target gene was inserted into the target site correctly.

3.2 Functional validation of the complete blue light-controlled system

After determining that the plasmid was correctly constructed and successfully transformed, we verified the light-sensitive function of the pSB1C3-stuffer-Lp plasmid (BBa_K4427002) in a dark room. With reference to the literature, we performed periodic experiments on engineered bacteria BL21 in a dark room with blue light irradiation for 2 hours and duck treatment for 2 hours alternatively, for a total of 8 hours, and a control experiment was conducted in complete darkness. The experimental results showed that the engineered bacteria containing the pSB1C3-stuffer-Lp plasmid (BBa_K4427002) successfully expressed 2-PE, which validated the effectiveness of our blue light-controlled system.

3.2.1 SDS-PAGE of crude enzyme solution

We performed SDS-PAGE experiments on the two groups of bacterial solution, obtained the following figure (Fig. 16). The molecular weight of Adh1 is 37 kDa and KdcA is 62 kDa, which is comparable to the molecular weight of the Marker corresponding to the bands in the electropherogram, which can tentatively prove that the pSB1C3-stuffer-Lp plasmid (BBa_K4427002) successfully expresses the target protein we need.

3.2.2 chromatograph characterisation of the end product phenylethanol (2-PE)

The above experiments demonstrated that E. coli BL21 transfected with pSB1C3-stuffer-Lp plasmid (BBa_K4427002) could catalyze the synthesis of the substrate phenylpyruvate into 2-PE. We then performed the final product expression experiments, and compared the samples with the standard 2-PE. The following results were obtained, identifying our samples as 2-PE.

The results of the experiment were as follows:

(1)Gas chromatogram of Standard 2-PE sample

(2)Gas chromatogram of the bacterial extract sample

2-PE cleavage mechanisms in electron bxombardment ion sources:

(3) Mass spectra of standard 2-PE samples

(4) Mass spectra of extracted 2-PE samples in bacterial broth

The results of gas chromatography and mass spectrometry showed that the bacteriophage extract samples contained 2-PE, indicating that the overall blue light expression system was successful. We also used liquid chromatography to roughly determine our 2-PE yields as follows:

3.3 Separate individual validation of KdcA and Adh1

To describe our process in more detail, we constructed two plasmids, pET-22b-Adh1 ( BBa_K4427007) and pET-30a-KdcA (BBa_K4427008), for two key enzymes in phenylethanol synthesis, ethanol dehydrogenase (Adh1) and α-keto acid decarboxylase (KdcA). And validated them individually as follows.

3.3.1 Nucleic acid electrophoresis, double digestion assay

To ensure that the Adh1 gene ( BBa_K4427004) and KdcA gene (KdcA BBa_K4427005) were inserted in the correct position and to verify that the plasmids were correctly transformed, we performed plasmid extraction, double digestion (XhoI and NdeI) and agarose gel electrophoresis on DH5α bacteria solution after transformed with the pET-22b-Adh1 ( BBa_K4427007) and pET-30a-KdcA (BBa_K4427008). The results showed that our plasmid was successfully transformed into DH5α successfully and our target gene was inserted into the target site correctly

3.3.2 SDS-PAGE of Adh1 and KdcA

(1)Adh1:

To initially determine whether the engineered bacteria expressed Adh1, we respectively performed SDS-PAGE of the proteins in BL21 before and after IPTG induction, and the supernatant and precipitate after induction of the crude extract. The results (Fig. 20)showed that the target protein Adh1 was present in all four samples at the same time, although initially most of the protein was expressed in the inclusion bodies, but after subsequent adjustment of the induction expression conditions,we obtained the desired protein in the supernatant finally.

(2) KdcA:

To initially determine whether the engineered bacteria expressed KdcA, we respectively performed SDS-PAGE of the proteins in BL21 before and after IPTG induction, and the supernatant and precipitate after induction of the crude extract. The results (Fig. 21) showed that the target protein KdcA was present in all four samples at the same time, and a high level of heteroproteins appeared. After mapping the conditions of Adh1 expression, we succeeded in obtaining a large amount of the desired target protein in the supernatant in the expression of KdcA.

3.3.3 Enzyme activity assay

Our aim was to ensure the enzymes synthesised by the engineered bacteria would complete the catalysis of the substrate and ultimately synthesise the odour substance we wanted. Therefore, we performed in vitro enzyme activity assays for the two key enzymes involved in the reaction, Adh1 and KdcA, respectively. However, considering the toxicity of phenylacetaldehyde, we decided to switch the substrates for the in vitro experiments to safer pyruvate and acetaldehyde for safety reasons.

(1) NADH absorption spectroscopy of Adh1

Adh1 from Saccharomyces cerevisiae reduces aldehyde groups to hydroxyl groups and converts NADH to NAD+ in order to maintain glycolysis. Both NADH and NAD+ have UV absorption peaks at 260 nm, but only NADH has a UV absorption peak at 340 nm. We used this property to check the absorbance of the reaction system at 340 nm to indirectly verify whether the reaction is taking place.

We used acetaldehyde and NADH as reaction substrates and Tris-HCl at pH = 7 as a buffer. The absorbance of the solution was measured using a UV spectrophotometer at reaction times of 0, 3, 6, 9, 12, 18 min to verify the catalytic effect of Adh1 on this reaction. The results (Fig. 22) showed that the absorbance of the system decreased with increasing reaction time when irradiated with UV light at a wavelength of 340nm, which proved that our enzyme was active.

(2) Enzyme concentration-dependent enzymatic activity assay for Adh1

After spectroscopic determination, we continued with the enzyme concentration-dependent enzymatic activity assay of Adh1 under 340 nm UV light. We carried out five sets of Adh1 gradient experiments at 3, 4, 4.5, 5 and 6 μL respectively. The experimental results showed that the enzyme solution volume at 3-6 μL showed an accelerated rate of substrate reduction with increasing enzyme concentration and a higher reaction rate.

(3) Absorption spectroscopic determination of NADH in the KdcA cascade reaction

KdcA from Saccharomyces cerevisiae catalyzes the conversion of alpha-keto acids to aldehydes, and the resulting aldehydes can participate in subsequent reactions catalyzed by Adh1. Therefore, we can verify the effectiveness of KdcA by showing that the entire cascade reaction is proceeding normally by the reaction of Adh1 with known enzyme activity.

We used pyruvic acid (simplest α-keto acid) as the reaction substrate, Tris-HCl at pH = 7 as a buffer. KdcA can catalyse the reaction of pyruvic acid to form acetaldehyde, and then we used Adh1 to catalyse the reaction of acetaldehyde to ethanol to determine the activity of KdcA. We used a UV spectrophotometer to measure the absorbance of the solution at reaction times of 1, 2, 3, 4 and 5 min. The results showed that NADH had a high absorbance when irradiated with UV light at a wavelength of 340nm and its content decreased with increasing reaction time, proving that our enzyme was active.

(4) Enzyme concentration-dependent enzymatic activity assay of KdcA

After spectroscopic measurements, we continued with the enzyme concentration-dependent enzymatic activity assay of KdcA under UV light at 340 nm. We added 10, 15, 20, 30, 35 and 40 μL of KdcA to the substrate and performed six sets of control experiments. The results showed that the rate of substrate reduction became increasingly faster with increasing enzyme concentration at enzyme solution volumes of 10-30 μL, the higher the enzyme activity. Anomalously, the reaction rate at 35 μL of enzyme solution slowed down compared to 30 μL, which was outside the confidence interval for the overall data trend, so we decided to discard this set of unreasonable data, which was tested by the Grubbs method as a suspect value that should be discarded.

3.3.4 Cascade catalytic activity of bacterial fluids.

We measured the activity of KdcA and Adh1 using pyruvate as the reaction substrate and a citric acid/sodium hydroxide solution at pH=6 as a buffer to verify the functionality of the pSB1C3-stuffer-LP plasmid (BBa_K4427002) by using a cascade reaction in which KdcA catalyzes the reaction of pyruvate to produce acetaldehyde and Adh1 then acetaldehyde catalyzes ethanol. We added 10, 15, 20, 30, 35 and 40 μL of the dual enzyme enzyme solution to the substrate and performed six sets of control experiments. The results (Fig. 25) showed the rate of substrate reduction was increasingly rapid with increasing enzyme concentration at 10-40 μl of enzyme solution. This is consistent with the results of the cascade reaction between KdcA and Adh1, which suggests that our expression system is effective.

4. The Suicide system

The suicide system consists of an initiation system and a virulence protein (blrA). The switch system consists of a red light-responding switch and a blue light-responding switch in parallel, and it is the simultaneous irradiation of red and blue light that initiates the expression of downstream virulence proteins. Due to time issues, the red light-responding switch, the blue light-responding switch and the toxin function of the suicidel system were verified separately.

4.1 Light switch for the suicide system

The initiation switch of the suicide system consists of a red light-responding switch and a blue light-responding switch in parallel, and it is the simultaneous irradiation of red and blue light that initiates the expression of downstream toxic proteins. Due to time issues, we only verified the blue light-responding switch and the red light-responding switch separately as described above, and the results showed that both switches were effective.

4.2 Functional validation of toxic proteins

We conducted a collaborative experiment with HainanU_China to validate the virulence proteins. They added 1 μL virulence gene plasmid to 50 μL competent cell, coated the plates after transformation according to the transformation process, added 50 μL solution to each plate and incubated them overnight at 37°C. The final result is shown here. The plate coated with the bacteria that have transformed the blrA has no colony, while the control group grows normally.