E N G I N E E R I N G
Engineering Success
Summary
"Kill switch" is defined as an artificial system that causes cell death under certain conditions. In other iGEM team projects, kill switch is usually used to ensure the biological safety of the project. In this project, our team designed death programs to program and control life and present a series of "engineering bacterial death simulators" in the form of installation art, in which bacteria will present their life performances.
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. The death of engineered bacteria warns people that if environmental damage is not prevented in time, our lives will also be negatively affected. We hope to warn everyone that we need to curb the deterioration of air pollution, ozone hole and other environmental problems through our efforts.
In Plan B, our T4 lysis device and beta-galactosidase synthesis pathway showed the effect of blue color reaction, 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. Macroscopically, it is a visual spectacle, while microcosmically, it is the doomsday aesthetics presented by innumerable cell deaths to reflect thoughts on bioethics.
Plan A: Death from UV pollution
In our design, we used UV-inducible switch to turn on the expression of lethal gene. After full exposure of sunlight, bacteria can synthesize toxins and die. We have referenced the design of UT-Tokyo iGEM team in 2011 and SZ-SHD team in 2020, and decided to adopt the UV-inducible switch, which is sulA promoter (BBa_K518010) proposed by iGEM11_UT-Tokyo (2011). And the lethal gene we chose is the relE toxin (BBa_K185047), an RNase that preferentially cleaves mRNAs bound to the ribosome at the second position of stop codons [1]. The expression of the relE gene has been shown to severely inhibit translation and prevent colony formation.
1. sulAp promoter testing
In plan A, we expected the engineered bacteria to turn on relE gene expression and die under UV induction. In order to achieve this goal, we first studied 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 (Gene circuit 1), and then transformed it into BL21.
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.
Fluorescence can be observed after UVC induction. Where there were 2-3 times increments in fluorescence than normal, and there was a positive correlation between the time of UVC induction and fluorescence intensity, as displayed in figure 1.
The experimental results prove that the sulAp promoter successfully triggered the expression of eGFP under UV induction.
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).
2. Expression of relE toxin gene
We then replaced the eGFP gene with relE toxin gene (Gene circuit 2) to test whether sulA promoter could induce the expression of relE.
Gene circuit 2: pSB1C3-sulAp-relE
Although the expression of relE gene results in bacterial growth slower than the control group, 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.
3. Use of Amp30E Amplification Device
We analyzed the results, and speculated that it might be caused by the low expression of relE gene. Therefore, we made improvements to the existing circuits based on literature research.
We applied a typical cellular sensor that can be abstracted as a three-stage processor comprising a sensing module that recognizes and transduces external signals into intracellular transcriptional signals, a computing module that modulates the transduced sensor signals, and an output actuating module that executes physiological responses.
We used the promoter of sulA as sensor module. The amplifier module (Amp30E Amplification Device) includes hrpR (BBa_K4226001), hrpS (BBa_K4226002) and PhrpL (BBa_K4226003) are engineered multi-layered transcriptional amplifiers that can be able to sequentially boost output expression level of target genes [2].The eGFP was used as an output module to test the effect of amplifier module.
In this part, we improved the parts: HrpR Gene (BBa_K1014001), HrpS Gene (BBa_K1014000) and Promoter hrpL (BBa_K1014002) uploaded by iGEM13_HIT-Harbin.
Gene circuits 3 was designed to test the effect of Amp30E using reporter gene eGFP:
The results showed that the Amp30E Amplification Device significantly increased the fluorescence expression of eGFP. This result greatly demonstrates the excellent capability of Amp30E Amplification Device as shown in Figure 3:
Figure 3: The result of eGFP expression (Fluorescence per OD) after irradiated under UVC (254nm). Competent host cell: BL21. Green fluorescence spectrum: 485/510nm. 0-8h: data collection time after UVC induction.
4. Amp30E increased the expression of relE
On this basis, we replaced the eGFP gene with relE toxin gene to test whether Amp30E system can increase the expression of relE gene. Gene circuit 4 was constructed as follows:
Gene circuit 4: pSB1C3-sulAp-Amp30E-relE
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). So far, we have achieved better results through reconstructing the gene circuits.
Figure 4: The OD600 of recombinant bacteria after irradiated under UVC (254nm). Competent host cell: DH10B. 0-8h: data collection time after UVC induction.
5. Verification of increased expression of relE
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).
The relE toxin gene and the mScarlet-I gene were fused to construct a fusion gene, in order to detect the amount of relE protein synthesis. We removed the terminator of relE and fused it to the mScarlet-I gene. And the 3WJ-Bro was used to ligate fluorescent aptamers to examine the relE RNA synthesis at transcriptional level (Gene circuit 5).
Gene circuit 5: pSB1C3-sulAp-Amp30E-relE-mScarlet-I-3WJ-Bro
RNA synthesis of relE:
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 5). The results indicate that the Amp30E Amplification Device significantly increased the RNA synthesis of relE gene as expected.
Figure 5: 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.
Protein synthesis of relE:
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 6), indicating the mScarlet-I correctly displayed the increasing tendency of relE protein synthesis under the effect of Amp30E Amplification Device:
Figure 6: 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.
Plan B: T4 lysis Device and beta-galactosidase synthesis
We designed another 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 [3,4].
According to the records of the team SZ-SHD (2020), the competent cells could died rapidly after the introduction of T4 Holins and lysozyme genes. Therefore, in our project, we first used the inducible pBad/araC promoter (BBa_I0500) to suppress expression, preventing the recombinant cells from dying immediately.
At the same time, we inserted the lacZ gene (BBa_I732019) in the T4 lysis Device (Gene circuit 6). The lacZ gene controls the synthesis of β-galactosidase, which can react with X-Gal in blue color [5]. This design provides great inspiration for the artistic design of this project.
As shown in figure 7, 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.
Figure 7: 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:
Figure 8: The lacZ gene controls the synthesis of β-galactosidase, which can react with X-Gal in blue color.
We used this experimental phenomenon to create a piece of artwork. Please click to see more details.
To sum up
These engineering cycles (design -> build -> test -> learn -> design...) greatly promoted the development of our project.
Both designs achieved great engineering success in synthetic biology!
For more details, please see the Parts page
References:
[1] Christensen S K, Mikkelsen M, Pedersen K, et al. RelE, a global inhibitor of translation, is activated during nutritional stress[J]. Proceedings of the National Academy of Sciences, 2001, 98(25): 14328-14333.
[2] Wan, X., Volpetti, F., Petrova, E. et al. Cascaded amplifying circuits enable ultrasensitive cellular sensors for toxic metals. Nat Chem Biol,2019, 15, 540-548.
[3] Ramanculov E, Young R. Functional analysis of the phage T4 holin in a λ context[J]. Molecular Genetics and Genomics, 2001, 265(2): 345-353.
[4] Young R, Bläsi U. Holins: form and function in bacteriophage lysis[J]. FEMS Microbiology Reviews, 1995, 17(1-2): 191-205.
[5] Diao W, Guo L, Ding Q, et al. Reprogramming microbial populations using a programmed lysis system to improve chemical production[J]. Nature Communications, 2021, 12(1):1-14.