P A R T
Part Collection
Basic Parts:
1.RelE toxin
The relE toxin (BBa_K185047) is an RNase that preferentially cleaves mRNAs bound to the ribosome at the second position of stop codons. Researchers found that relE inhibits protein synthesis by cleaving mRNA codons in a sequence specific way with preference for the stop codon UAG [1]. As a result, expression of the relE gene has been shown to severely inhibit translation and prevent colony formation. Our project plans to use ultraviolet light to induce the sulA promoter to initiate the expression of relE toxin protein, so as to achieve the effect of bacterial death.
2.sulA promoter
SulA is responsible for stress-induced halt of cell division. The promoter of sulA, sulAp (BBa_K518010), is induced by various stress factors, including ultraviolet irradiation [2]. In our design, the sulAp was evaluated with the aid of eGFP gene, coded for a protein with a fluorescence spectrum of 485/510nm. We constructed the circuit below and transformed into the E. Coli strain BL21 (DE3).
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 as follows:
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).
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 irradiation and fluorescence intensity, as displayed in the diagram above.
Composite Part:
1.T4 lysis Device and beta-galactosidase synthesis
T4 lysis Device
T4 lysis Device is a system derived from bacteriophage T4, which has two main components: T4 holin (BBa_K112805) and T4 endolysin (BBa_K112806). Holins from T4 bacteriophage assemble together to form pores on inner membrane of bacteria allowing lysozyme to reach periplasm and degrade peptidoglycan layer. T4 endolysin is the lysozyme from enterobacteria phage T4 degrades peptidoglycan layer. The lysozyme is expected to be able to reach the peptidoglycan layer through holins, which degrades the plasma membrane to introduce cellular contents into the periplasmic space, causing the bacteria to rupture and die [3].
According to the records of the team SZ-SHD (2020), the competent cells 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. We recombined the pBAD promoter and T4 lysis Device with pSB1C3 vector. The OD600 of the recombinant bacteria decreased significantly after induction with different concentrations of arabinose. The results are as follows:
Figure 2: 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).
As shown in the figure above, the OD600 of recombinant DH10B cells with pBAD-lacZ-T4 lysis gene circuits reduced significantly by 2-3 times than non-recombinant cells after induced by arabinose.
Beta-galactosidase synthesis
At the same time, we inserted the lacZ gene (BBa_I732019) in the T4 lysis Device genetic circuit. The lacZ gene controls the synthesis of β-galactosidase, which can react with X-Gal in blue color, the effect is shown below:
2.Amp30E Amplification Device
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 [4]. We first 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), and the eGFP was used as an output module to test the effect of amplifier module.
We recombined the sulAp, Amp30E and eGFP with pSB1C3 vector. The recombinant cells were cultured to optimum cell growth (OD600) and irradiated under ultraviolet C (UVC, 254nm) . The parameters were then measured using microplate reader for 8 hours. The fluorescence per OD significantly increased after induction with UVC exposure. The results are as follows:
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.
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.
We use this amplification device to increase the cytotoxic effect of relE toxin on cells. The genetic circuit that contains sulAp, Amp30E and relE gene was constructed as follows:
As expected, the OD600 of the recombinant bacteria (pSB1C3-sulAp-relE) decreased after UVC induction, and the Amp30E Amplification Device significantly increased the inhibitory effect of relE on bacterial growth as shown below:
Figure 4: The OD600 of recombinant bacteria after irradiated under UVC (254nm). Competent host cell: DH10B. 0-8h: data collection time after UVC induction.
3.RNA and protein reporting systems
3WJ-Bro (BBa_K4226000) and mScarlet-I (BBa_K3977002) were used as RNA and protein reporting systems in the experimental design. Firstly, we used a protein- independent reporter system, 3WJ-Bro, based on a fluorescent RNA aptamer. 3WJ-Bro can be applied as a gene marker in creating a system for reporting the presence and expression of target gene [5], which is relE toxin gene at the transcriptional level in our project. Our results show that 3WJ-Bro can report the transient expression of relE gene in Escherichia coli cells at the transcriptional level, obviating the need for accumulation of foreign proteins.
mScarlet-I is a truly monomeric red fluorescent protein with record brightness, quantum yield (70%) and fluorescence lifetime (3.9 ns) [6], ideal for use as a fluorescent marker. 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 at transcriptional level. So, we have designed the genetic circuit that contains sulAp, Amp30E, relE gene, mScarlet-I and 3WJ-Bro, and transferred it into DH10B competent cells
After bacterial culture and UV induction, we measured OD600 and fluorescence values under 485/510nm and 579/616nm respectively. Then, the fluorescence per OD was calculated. According to the results, the reporter system includes 3WJ-Bro and mScarlet could successfully reflect the expression level of relE protein as shown in figure 5 and 6.
The curve of experimental group was significantly higher than that of control group and the amount of relE RNA synthesis increased with incubation time (Figure 5). The results indicate that the Amp30E Amplification Device significantly increased the RNA synthesis of relE gene as we 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.
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.
To summarize the results above, the parts of 3WJ-Bro and mScarlet-I were available for reporting systems and showed the expected trend of relE synthesis in the experimental design.
Parts tables:
1. Basic Parts
| Numbers | Parts Name | Parts Number | Types | Length（bp） |
|---|---|---|---|---|
| 1 | RelE toxin | BBa_K185047 | Coding | 360 |
| 2 | sulA promoter | BBa_K518010 | Regulatory sulAp | 52 |
| 3 | T4 holin | BBa_K112805 | Coding | 676 |
| 4 | T4 endolysin | BBa_K112806 | Coding | 514 |
| 5 | lacZ | BBa_I732019 | Generator | 3230 |
| 6 | mSarlet-I | BBa_K3977002 | Reporter | 693 |
| 7 | RBS | BBa_B0034 | RBS | 12 |
| 8 | Double terminator | BBa_B0015 | Terminator | 129 |
| 9 | Inducible pBad/araC promoter | BBa_I0500 | Regulatory sulAp | 1210 |
2. Basic New Parts（Uploaded by CAFA_China 2022）
| Numbers | Parts Name | Parts Number | Types | Length（bp） |
|---|---|---|---|---|
| 1 | 3WJ-Bro | BBa_K4226000 | Reporter | 105 |
| 2 | hrpR | BBa_K4226001 | Coding | 945 |
| 3 | hrpS | BBa_K4226002 | Coding | 909 |
| 4 | hrpL Promoter | BBa_K4226003 | Regulatory | 208 |
3. Composite New Parts（Uploaded by CAFA_China 2022）
| Numbers | Parts Name | Parts Number | Types | Length（bp） |
|---|---|---|---|---|
| 1 | Amp30E Amplification Device | BBa_K4226004 | Device | 2271 |
| 2 | RNA and protein reporting systems | BBa_K4226005 | Reporter | 806 |