Overview

Our experiment consists of the functional validation of two systems, the blue-light system and the red-light system. The main body of our experiment is divided into 3 aspects: the blue-light system to release 2-PE (phenylethanol), the red-light system to release 3-oxo-C12-HSL, and the simultaneous activation of both systems to kill the engineered E. coli.

For the blue-light pathway, we selected the EL222 protein, which could dimerize to bind to the PBLind promoter, initiating the expression of the downstream gene expression under blue light[1]. In contrast, for the red-light pathway, we used BphP1 and PpsR2 as a photosensitive system, which could be activated by red light to initiate downstream gene expression[2][3].

For the selection of target gene, we focused on 2-PE. As a terpenoid, 2-PE has been shown to be beneficial in relieving sadness with its distinctive floral scent, and this mood-relieving effect can be scientifically confirmed by the detection of brain waves at sleep[4]. Past researches have reported the successful expression of 2-PE in E.coli . We need to use its natural production and transfer the genes downstream to build an Ehrlich pathway in E.coli to achieve efficient expression of 2-PE[5]. This ensures that the selected target gene, 2-PE, can be expressed properly in E.coli. In addition, we associate the target gene with EL222, so that we can use blue light to control the expression of the target gene.

In consideration of the biosafety of the project, we proposed to use both red and blue light together to kill the engineered bacteria in case of bio-contamination. This idea was achieved by producing an inducer 3-oxo-C12-HSL under red light, and opening AND Gate channels with the inducible substance when irradiated with blue light to induce the expression of downstream virulent protein genes[1]. Having the product expressed inside the cell was not the end of the story, instead, they should be released. Fortunately, as small molecules, 2-PE had innate properties that allowed it to cross the cell membrane along the concentration gradient. Moreover, as an alcohol, 2-PE could diffuse out of E. coli more easily due to its high fat-solubility[5].

1. introduction

① Plasmid construction

We plan to realize the function of producing phenylethanol under the control of blue light[6], so we designed the plasmid pSB1C3-stuffer-all (BBa_K4427012).

However, we found that the construction of such a huge plasmid was not only difficult, but also the actual transformation and expression effect was not satisfactory. Therefore, we properly simplified the plasmid, removed some non critical enzymes, and temporarily removed the toxic protein gene blra from the whole plasmid. Therefore, our simplified plasmid pSB1C3-stuffer-LP (BBa_K4427002) came out.

In order to better explore whether the function of each component can be realized normally, we built several sets of plasmids separately for several components in the plasmid pSB1C3-stuffer-LP (BBa_K4427002) to verify its function.

We constructed a plasmid pSB1C3-stuffer-pro (BBa_K4427009) to independently verify the function of photosensitive protein EL222. By adding the fluorescent protein gene mrfp1, we can directly judge whether the function is normal by the presence or absence of fluorescence.

In order to investigate whether the two key enzymes Adh1 and KdcA related to phenylethanol production are normal, we constructed plasmids pET-22b-Adh1 (BBa_K4427007) and pET-30a-KdcA (BBa_K4427008) respectively. These two plasmids can express Adh1 and KdcA separately, so that we can better study the properties of these two enzymes in vitro.

Similar to the blue light system, we also independently verified each part of the red light system.

First, we designed the plasmid pET-22b-RLP (BBa_K4427016) to independently verify the function of the red light starting system. By adding the fluorescent protein sfGFP gene, we can directly judge whether the function is normal by the presence or absence of fluorescence.

We originally planned to carry out red and blue light co adjustment experiments to verify whether the function of the AND gated channel was normal. Unfortunately, due to time problems, we could only save this verification for a later time.

② E.coli selection

In order to balance the accuracy of the experimental results and the stability of the experimental process, we decided to choose E.coli BL21 as the experimental strain after comparing the normal curves and product yield of E.coli DH5α and BL21.

2. Blue Light System

To ensure the accuracy and reliability of the blue light system, we used a separate validation format, transforming plasmids of each key protein in the system (EL222, Adh1, KdcA) into E.coli separately and verifying their expression as well as their function.

① EL222（BBa_K4427003）

We first transformed pSB1C3-stuffer-pro (BBa_K4427009)into E.coli and first confirmed the successful transformation of the plasmid into E.coli by gel electrolysis, (note that thereafter all experimental manipulations were performed under shade) adding the EL222 expression inducer to the E.coli culture media to get the system started.

② KdcA（ BBa_K4427008 ）

KdcA (branched-chain alpha-ketoacid decarboxylase) is a ketoacid decarboxylase that converts phenylalanine PPA to PAD, which in turn converts PAD to 2-PE.

③ Adh1（ BBa_K4427007）

Adh1 (alcohol dehydrogenase) converts the PAD mentioned above catalyzed by KdcA into the final product 2-PE. This geotactic process uses the genetically catalyzed product PAD of the former enzyme, which is then converted to the final product 2-PE by Adh1.

3. Red Light system

To ensure the accurate reliability of the red light system, we used separate validation, i.e. transforming plasmids of each key protein in the system into E. coli separately to characterize their expression as well as their function.

BphP1 (BBa_K4427000), PpsR2 (BBa_K4427001) and Ho1 (BBa_K4427010)

We exploited this system of reversible light-induced binding between the bacterial phytochrome BphP1 and its natural partner PpsR2. In this system, Ho1 first 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[3]. For the red light part, we also used a block validation approach to demonstrate the proper functioning of the red light system.

4. Suicide system

In order to avoid unnecessary nutrient depletion of other flora caused by E.coli entering the decay phase, and also so that the process of gas synthesis and release can be effectively controlled to achieve an immediate on/off effect, we cleverly used the existing blue and red light pathways and added the E. coli virulence protein BlrA used by the iGEM team at XMU in 2021 downstream of the pathway intersection, and made a logical breakthrough to verify its function of causing the death of E. coli.

Our in-depth partner HNU_China helped us to complete the functional verification of the suicide system. As for the hardware design, we still adopted the mode of blue light intermittent irradiation to activate EL222 expression. In other words, the alternate treatment of 2 hours of irradiation followed by 2 hours of darkness to the engineered bacteria (to reduce the toxic effect of blue light irradiation on the bacteria, and at the same time to shift the chemical balance) continued for 8 hours in total. Subsequently, the bacteria lysed and died due to the large accumulation of BlrA toxic protein in E.Coli.

① blrA （BBa_K4427028）

Firstly, while the blue light system was normally expressed, we have added a "small tail" at the end of the pathway—the LasI promoter—which, while not activated, prevented the normal production of the virulence protein BlrA and kept the bacteria active to synthesize and release odour molecules. Once the AND gate opens, the LasI promoter will be activated and will transcribe a large amount of BlrA toxin, which causes the bacterium to autolysis and die, thus terminating the synthesis and release of odour molecules.

② HSL

As mentioned above for logic gap, when would the AND logic gate turn on? We designed the normal irradiation of red light to produce 3-oxo-C12-HSL, which can open the AND gate channel thereby initiating the expression of the downstream toxic protein blrA.

5. References

[1] Jayaraman P, Devarajan K, Chua T K, etal. Blue light-mediated transcriptional activation and repression of gene expression in bacteria[J]. Nucleic Acids Research, 2016, 44(14):6994-7005.

[2] Baumschlager A, Khammash M. Synthetic Biological Approaches for Optogenetics and Tools for Transcriptional Light-Control in Bacteria[J]. Advanced Biology, 2021, 5(5).

[3] Ong N.T, Olson E.J, Tabor J.J. Engineering an E. coli Near-Infrared Light Sensor[J]. ACS Synthetic Biology, 2019(7):240-248.

[4] Herz RS. Aromatherapy facts and fictions: a scientific analysis of olfactory effects on mood, physiology and behavior. Int J Neurosci. 2009;119(2):263-90.

[5] Liu, C., Zhang, K., Cao, W. et al. Genome mining of 2-phenylethanol biosynthetic genes from Enterobacter sp. CGMCC 5087 and heterologous overproduction in Escherichia coli.[J] Biotechnol Biofuels.

[6] Schwaiger K N, Voit A, Hana Dobiašová, etal. Plasmid Design for Tunable Two-Enzyme Co-Expression Promotes Whole-Cell Production of Cellobiose[J]. Biotechnology Journal, 2020, 15(11).