Device 1: Detection and Degradation Circuit
Our first device consists of a stationary-phase inducible promoter that is activated in the presence of IPTG. To ensure that the construct responds only to RDX, we included an RDX-specific riboswitch upstream of each gene. This riboswitch allows us to control the translation of the genetic construct. In the presence of RDX, the genes xplA and xplB are expressed. These genes function together as a complex to degrade RDX: xplB transfers electrons from NADPH to activate the catalytic center of xplA (Flavodoxin domain), which then catalyzes the reductive denitrification of RDX. Finally, our reporter gene, mCherry, a Red Fluorescent Protein with an emission peak at 610 nm, serves as an indicator of RDX detection and successful translation of the construct.
Figure 1. SBOL representation of Device 1, including the σ38 stationary-phase inducible promoter, RDX riboswitch, degradation genes (xplA, xplB), and the reporter gene (mCherry).
Table 1. Genetic parts of Device 1
| BioBrick | Type | Part Name | Function and Usage |
|---|---|---|---|
| BBa_K086030 | Promoter | σ38 stationary phase inducible promoter | This promoter is adapted from the Lutz-Bujard LacO promoter (BBa_R0011) by IIT Madras in 2008. It contains the sigma factor 38 (σ38), which is responsible for the transcription of genes under stress conditions [1]. It also includes a LacO operon, making the presence of IPTG necessary to initiate transcription. This design provides tighter control over gene expression. |
| BBa_K5252007 | Riboswitch | RDX Riboswitch | This riboswitch, developed by [2], was selected from a large aptamer library using the SELEX method (Systematic Evolution of Ligands by Exponential Enrichment). Among the candidates, clones 4 and 11 exhibited the highest activation ratios (11-fold and 5-fold, respectively), characterized by low basal fluorescence and strong induction upon RDX exposure. The riboswitch regulates gene expression at the translational level, which is critical for our project as it requires a riboswitch between each gene in a multi-cistronic construct. |
| BBa_K3857002 | CDS | xplB | *The xplB gene encodes for a partner Flavodoxin Reductase which is complexed with xplA. This reductase promotes the activation of the catalytic center of xplA via electron transfer from NADPH to a Flavodoxin domain fused to the N-terminal of the P450 domain of xplA [3]. This allows xplA to catalyze the reductive denitration of explosive organic contaminant, hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and ring cleavage under aerobic and anaerobic conditions. Even though it is not required, xplB contributes to RDX catabolism, reinforcing the already efficient activity of xplA. Some bacteria have shown that the absence of xplB (with an existing xplA in the system) reduced the rate of RDX degradation by 70% [3]. |
| BBa_K3670004 | CDS | xplA | *The xplA gene encodes for the Flavodoxin domain fused (at the N-terminus) of a P450 cytochrome. Studies demonstrate unequivocally that his gene product is necessary for RDX degradation [3], especially in the cases where bacterial species do not have natural RDX-degrading systems. Depending on anaerobic or aerobic conditions, RDX converts to MEDINA and 4-nitro-2,4, diazabutana (NDAB) respectively. In either condition, nitrite and formaldehyde will form as byproducts. |
| BBa_K592010 | Protein Domain | amilGFP | *AmilGFP is a chromoprotein derived from the coral Acropora millepora, which naturally exhibits a readily visible, strong yellow color when expressed. It encodes for a small peptide functioning as a degradation tag that will allow for fine-tuning protein levels and thus regulating the GFP in the bacteria. |
| BBa_J176005 | Protein Domain | mCherry | In certain experiments, we employ mCherry rather than amilGFP, as AmilGFP is difficult to detect in culture media. *In the presence of RDX, the reporter gene emission of the Red Fluorescent Protein can be quantified to analyze the transcription of the first device. The activity of the first device can be measured by analyzing the correlation of the intensity of color emission while measuring the concentrations of RDX. The mCherry gene expresses a red light that has emission at 610nm and excitation at 597nm. |
| BBa_B0010 | Terminator | rrnB T1 terminator | *The terminator is a sequence inserted to halt the transcription of our first device, based on the BioBrick terminator. |
* Information retrieved from the iGEM-RUM UPRM 2022 wiki
Supplementary Device 1: Detection and Degradation Circuit under Heat Shock Conditions
As part of our project, we are committed not only to solving the problem of RDX degradation, but also to ensuring biosafety and protecting the environment. When XplA and XplB degrade RDX via reductive denitrification, formaldehyde and nitrite are formed as byproducts. Since these compounds are highly carcinogenic, their presence poses a significant risk to human health [4]. For this reason, all of our biological processes will be contained within a bioreactor. To gain greater control over the activation of our genetic device, we plan to introduce a heat-shock inducible promoter that can be activated in the presence of IPTG. Heat shock is easier to regulate within a bioreactor, making it a practical control mechanism for our system.
Figure 2. SBOL representation of Device 1, including the σ32 heat shock inducible promoter, RDX riboswitch, degradation genes (xplA, xplB), and the reporter gene (mCherry).
Table 2. Genetic parts of Supplementary Device 1
| BioBrick | Type | Part Name | Function and Usage |
|---|---|---|---|
| BBa_K086030 | Promoter | σ32 stationary phase inducible promoter | This promoter is adapted from the Lutz-Bujard LacO promoter (BBa_R0011) by IIT Madras in 2008. It contains the sigma factor 38 (σ38), which is responsible for the transcription of genes under stress conditions [1]. It also includes a LacO operon, making the presence of IPTG necessary to initiate transcription. This design provides tighter control over gene expression. |
Device 2: Killswitch
Our third device is an AND gate designed to function as a kill switch, activated in the presence of both formaldehyde and nitrite, which are byproducts of the first device. This circuit consists of two inducible promoters: PyeaR, which is activated by nitrite, nitrate, or nitric oxide under both aerobic and anaerobic conditions, and Pfirm, which is activated by formaldehyde. Downstream of these promoters are supD and t7ptag, which work together to activate the T7 promoter. The t7ptag sequence encodes a T7 polymerase containing two amber mutations that would normally prevent its translation. However, supD encodes a tRNA amber suppressor that eliminates these mutations, allowing proper expression of the polymerase. Once expressed, the T7 polymerase drives the activation of the T7 promoter, leading to the production of Colicin. Colicin induces bacterial lysis through its endonuclease activity, thereby halting detection and biodegradation of RDX by killing the engineered bacteria and ensuring biosafety.
Figure 3. SBOL representation of Device 2.
Table 3. Genetic parts of Device 2
| BioBrick | Type | Part Name | Function and Usage |
|---|---|---|---|
| BBa_K216005 | Regulatory | PyeaR promoter | This inducible promoter will be activated in the presence of nitrate, nitric oxide or nitrite. Once nitrite and nitrate enter Escherichia coli, they will be converted into nitric oxide. The transformation to nitric oxide occurs so that this promoter can be inactivated, and transcription of unwanted genes does not proceed. Unlike other E. coli promoters responding to nitrate and nitrite, this promoter is not repressed under aerobic conditions. In other words, PyeaR works successfully in both anaerobic and aerobic conditions. |
| BBa_K2728001 | Regulatory | Formaldehyde-Inducible Promoter pfirm | This inducible promoter is engineered to activate in the presence of formaldehyde. |
| BBa_K228100 | Composite | supD + terminator | supD produces a tRNA amber mutation suppressor that activates the mRNA produced by t7ptag. It can be well terminated by the terminator BBa_B0015. They are often fused after a certain promoter, in this case the Formaldehyde-Inducible Promoter pfirm. |
| BBa_K228000 | CDS | t7ptag (T7 polymerase with amber mutation) | t7ptag is a coding sequence that encodes for a T7 RNA polymerase with two amber mutations. For a successful translation of this gene’s mRNA to take place, an Amber mutation suppressor, supD, must be available to eliminate these mutations. t7ptag, as well as supD, is what makes the third device function as an AND gate. |
| BBa_I712074 | Regulatory | T7 promoter | T7 promoters work with T7 RNA Polymerase. This promoter activates in the presence of t7ptag. Activation of this gene expression will result in production of Colicin. |
| BBa_K117000 | CDS | lysis gene | The lysis gene encodes for the Lysis Protein in bacteria strains that produce Colicin. It activates the endonuclease activity of Colicin, killing our bacteria after biodegrading RDX. |
| BBa_B0010 | Terminator | rrnB T1 terminator | The terminator is a sequence inserted to halt the transcription of our first device, based on the BioBrick terminator. |
Information of device 2 retrieved from the iGEM-RUM UPRM 2022 wiki
References
[1] A. Shiratsuchi, N. Shimamoto, M. Nitta, T. Q. Tuan, A. Firdausi, M. Gawasawa, K. Yamamoto, A. Ishihama, and Y. Nakanishi, "Role for σ38 in prolonged survival of Escherichia coli in Drosophila melanogaster," J. Immunol., vol. 192, no. 2, pp. 666–675, Jan. 2014, doi: 10.4049/jimmunol.1300968. Available: https://pubmed.ncbi.nlm.nih.gov/24337747/
[2] J. O. Eberly, M. L. Mayo, M. R. Carr, F. H. Crocker, and K. J. Indest, “Detection of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) with a microbial sensor,” J. Gen. Appl. Microbiol., vol. 65, no. 3, pp. 145–150, 2019, doi: 10.2323/jgam.2018.08.001. Available: https://doi.org/10.2323/jgam.2018.08.001.
[3] C. S. Chong, D. K. Sabir, A. Lorenz, C. Bontemps, P. Andeer, D. A. Stahl, S. E. Strand, E. L. Rylott, and N. C. Bruce, "Analysis of the xplAB-containing gene cluster involved in the bacterial degradation of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine," Appl. Environ. Microbiol., vol. 80, no. 21, pp. 6601–6610, Oct. 2014, doi: 10.1128/AEM.01818-14. Available: https://journals.asm.org/doi/10.1128/aem.01818-14
[4] H. Ye, Y. Yang, L. Jiang, T. Zhe, J. Xu, and L. Zeng, "An intelligent sensing platform for discrimination of formaldehyde and nitrite in food," Chin. Chem. Lett., in press, Corrected Proof, available online Jan. 10, 2025, doi: 10.1016/j.cclet.2025.110840. Available: https://www.sciencedirect.com/science/article/abs/pii/S1001841725000270
