Proof of Concept
1.Design concept
We designed two genetic circuits to provoke in-depth thinking about environmental conservation from the perspective of art and synthetic biology.
In Plan A, we designed a UV-inducible switch to simulate the impact of excessive UV radiation from the ozone layer hole caused by complex human activities and atmospheric chemical process. After full exposure to sunlight, bacteria can synthesize toxins and die. The death of engineered bacteria warns people that if environmental damage is not prevented in time, our lives will also be negatively affected.
In Plan B, our T4 lysis Device and beta-galactosidase synthesis pathway showed the effect of blue color, symbolizing the tears of Horus' eyes in art. The engineered bacteria died and the solution turned blue. The mixture of solutions slowly dripped from the container, as if the tears of giant's eyes fell from the sky. Weeping for the death of bacteria caused by enhanced ultraviolet radiation caused by the ozone layer hole.
We hope to use our design concept to warn everyone that we need to curb the deterioration of air pollution, ozone hole and other environmental problems through our efforts.
Plan A: Death from UV pollution
In our design, we used UV-inducible switch (sulAp promoter) to turn on the expression of lethal gene (relE toxin gene).
1.1 sulAp promoter testing
We tested the activity of sulAp with and without UV irradiation with the help of eGFP gene. We constructed the circuit containing sulAp and eGFP with the vector pSB1C3.
Figure 1: The result of eGFP expression (Fluorescence per OD) after irradiated under UVC (254nm). Green fluorescence spectrum: 485/510nm. (eGFP -UV-: non-recombinant BL21 under no UV irradiation; eGFP -UV+: non-recombinant BL21 under UV irradiation for 1 min; 0 min: recombinant BL21 without UV irradiation; 0.5 min: irradiated under UV for 0.5 min; 1 min: irradiated under UV for 1 min and so on in a similar fashion). We then used sulAp promoter to turn on the expression of relE toxin. The effect of relE was not obvious as we expected, which is shown in figure 2. We still hope to obtain a better effect on inhibiting bacterial growth. Figure 2: The OD600 of recombinant bacteria after irradiated under UVC (254nm). Competent host cell: DH10B. 0-11h: data collection time after UVC induction. The establishment of mathematical models can help us describe the relationship between different parameters and variables in the project process by using a set of mathematical equations.In our project, UV-inducible promoters control the expression of GFP and toxin proteins, and we use mathematical models to describe the relationship between incubation time and fluorescence intensity under different durations of UV-induced induction.
Ultraviolet irradiation will increase the expression of fluorescent proteins per unit, making the overall fluorescence intensity stronger, but at the same time, the accumulation of toxin proteins in bacteria will also lead to a decrease in bacterial biomass (OD value) and weaken the overall fluorescence intensity.Through the whole model, we can know the change of fluorescence intensity under different UV irradiation time and incubation time.The model parameters are obtained by fitting our experimental data, and referring to some literatures and the work of other iGEM teams.Our assay can help us better understand the relationship between bacterial death and fluorescence intensity within the assay.
In our experiments, we assume: 1. The resources in the strained cultural environment are limited, and the population cannot grow infinitely.2. After receiving UV irradiation, GFP and RelE under the control of UV-sensitive promoter start to express at the same time.
According to the hypothesis, we set up the model and solved it. It can be known that in the absence of toxic proteins, when given different times of ultraviolet light , the fluorescence intensity emitted by bacteria cultured for the same time is related to the ultraviolet light.In the presence of toxic proteins, the growth of the strain conforms to the logistic curve. Since both RelE and GFP are under the control of the same UV-sensitive promoter element, the survival pressure brought about by the expression of toxic proteins is positively correlated with their protein expression levels. The protein expression is related to UV exposure time.
The software part is made based on experimental data and mathematical models. We visualize the experimental data through artistic processing, and explain the mathematical logic and experimental conclusions that are difficult for ordinary people to understand through wonderful and interesting artistic images, so that people are more interested in understanding synthetic biology, and also combine art and science to achieve cross-fertilization between disciplines. We applied Amp30E Amplification Device to increase the expression of relE gene. As we expected, the OD600 of the recombinant bacteria decreased after UVC induction (pSB1C3-sulAp-relE), and the Amp30E Amplification Device significantly increased the inhibitory effect of relE on bacterial growth (pSB1C3-sulAp-Amp30E-relE).
Figure 3: The OD600 of recombinant bacteria after irradiated under UVC (254nm). Competent host cell: DH10B. 0-8h: data collection time after UVC induction. In order to further confirm the phenomenon of increased expression of relE at transcriptional level, we introduced RNA and protein reporting systems including 3WJ-Bro (BBa_K4226000) and mScarlet-I (BBa_K3977002).
After bacterial culture and UV induction, we measured OD600 and fluorescence values under 485/510nm. RNA synthesis of relE was almost undetectable in circuits without Amp30E. The curve of experimental group was significantly higher than that of control group and the amount of relE RNA synthesis gradually increased with incubation time (Figure 4). The results indicate that the Amp30E Amplification Device significantly increased the RNA synthesis of relE gene as expected.
Figure 4: 3WJ-Bro was ligated with fluorescent aptamers in order to examine the level of relE RNA synthesis. Competent host cell: DH10B. Green fluorescence spectrum: 485/510nm. 0-8h: data collection time after UVC induction. Then, we measured OD600 and fluorescence values under 579/616nm to examine the protein synthesis of relE. The curve of experimental group was also higher than that of control group (Figure 5), indicating the mScarlet-I correctly displayed the increasing tendency of relE protein synthesis under the effect of Amp30E Amplification Device.
Figure 5: The mScarlet-I gene was fused with relE gene to detect the amount of relE protein synthesis. Competent host cell: DH10B. Fluorescence spectrum: 579/616nm. 0-8h: data collection time after UVC induction. We designed a suicide pathway based on T4 lysis Device, a system derived from bacteriophage T4, which has two main components: T4 holin (BBa_K112805) and T4 endolysin (BBa_K112806), that cause the bacteria to rupture and die. We used the arabinose inducible pBad/araC as promoter (BBa_I0500) and inserted the lacZ gene (BBa_I732019) in the T4 lysis Device. The lacZ gene controls the synthesis of β-galactosidase, which can react with X-Gal in blue color. This design provides great inspiration for the artistic design of this project.
In the proof of concept, the OD600 of recombinant cells with pBAD-lacZ-T4 lysis gene circuit reduced significantly by 2-3 times than non-recombinant cells after induced by different concentrations of arabinose. The concentration of arabinose was positively correlated with the degree of bacterial death according to the result.
Figure 6: The result of T4 lysis Device after induced by different concentrations of arabinose. (pSB1C3: non-recombinant DH10B without arabinose induction; pSB1C3-pBAD-lacZ-T4 lysis: recombinant DH10B without arabinose induction and with different concentrations of arabinose). In order to test the effect of lacZ gene and Beta-galactosidase synthesis, the recombinant cells (pSB1C3-pBAD-lacZ-T4 lysis) were placed in M9 medium and cultured for 12 hours. Subsequently, x-gal was added to the M9 medium, and the recombinant cells were induced by arabinose. The experimental phenomena are as follows. At this point, we have the desired effect at the experimental level, providing possibilities for the production of artwork: Figure 7: The lacZ gene controls the synthesis of β-galactosidase, which can react with X-Gal in blue color. In the meantime, we tried to make the blue solution appear in different gradation of color by using different concentrations of arabinose inducer. It can be seen that different colors are presented at different concentrations of inducers as follows: Figure 8: After induction of different concentrations of arabinose (0mM-225mM), the solution produced blue differences. In this way, we can change the gradation of the blue solution in our exhibition works just like a natural palette, which may make the audience have more interactions with the artwork. If we have more time for experiments, we will also test the amount of beta-galactosidase synthesis induced by different concentrations of arabinose using beta-galactosidase assay kit. We will continue to optimize our iGEM project in the future. On the one hand, we use artworks to popularize the knowledge of synthetic biology from the superficial to the deep for the public of different ages.For this reason, we have joined with Tsinghua and Peking University's experimental programs to visualize them and hold the TAP exhibition; on the other hand, a lot of artwork can evoke the audience reflecting on society, and we attempt to use artworks to evoke the public to reflect on synthetic bioscience, especially the conceptual issues such as moral ethics and philosophy that it brings.We therefore visualized the experimental lines, combining them with a classic artistic iconography theme.In addition, we explored how good art and technology combined products are formed, for example, how technology and art are combined in technology art works, what kind of survival paths do technology art companies find, etc. We try to integrate art with bioethics and environmental conservation issues that are hotly debated in the world. The core experiments and social activities are designed around these questions. We discussed bioethics in depth with other teams and a professor from Tsinghua and decided to design bacterial suicide pathways, in order to prevent genetic contamination.
We have, through careful selection of parts and promoter, raised the question of the ozone layer hole and the massive damage to many biological communities caused by ultraviolet enhancement. If we don't act timely, tears will drop from Horus 'Eye in a deteriorating environment on our planet. Please click to see more details about the implementation page. More details about experimental design were shown on the engineering page.
We cultured the recombinant bacteria in the dark to optimum cell growth (OD600) and irradiated them under ultraviolet C (UVC, 254nm) with an intensity of 15mWcm-2 for different periods of time. Then, each sample was cultured in incubating-shaker in darkness for 8 hours during which the fluorescence per OD was calculated.
The experimental results prove that the sulAp promoter successfully triggered the expression of eGFP under UV induction. 
1.2 Expression of relE toxin gene
1.3 concept of model
1.4 concept of software
1.5 Amp30E increased the expression of relE
1.6 Verification of increased expression of relE
RNA synthesis of relE:
Protein synthesis of relE:
Plan B: Concept of the Tears of Horus' Eye
2. Synthetic biology and art
3. Bioethics and environmental conservation