Parts
In this page you will find the utilized parts of each device in our project design.
Device 1: Detection Circuit
Our first device consists of a promoter fusion between hmp and hcp E.coli genes that would function in the presence of RDX. The luxI gene is an autoinducer synthase for AHL that will bind to the LuxR protein produced by the luxR gene. LuxR protein and AHL will then form a complex and bind to luxpr to activate its transcription in our second device. Between each gene a strong ribosomal binding site was added to control the efficient and accurate initiation of translation. Additionally, we used a degradation tag, LVA-ssra, after the luxR gene that would induce protein degradation and reduce the protein half-life so that accumulation of the protein would not accidentally activate our second device in the absence of RDX. A similar degradation tag is already integrated in the luxI gene sequence. Then our reporter gene mCherry, a red fluorescence protein with light emission at 610 nm, would help us know if transcription occurred by analyzing the intensity of color emission and the concentrations of RDX. Lastly, we used a terminator to stop the transcription of our device.
Figure 1. SBOL representation of Modified Device 1 genetic circuit.
Table 1. Parts of Modified Device 1
| BioBrick | Type | Part Name | Function and Usage |
|---|---|---|---|
| not available | Promoter | hmp/hcp fusion promoter | The enzyme hmp catalyzes the conversion of nitric oxide (NO) to nitrate (NO−3). The enzyme hcp is a high-affinity nitric oxide reductase. By developing a tandem of both the promoters of hmp and hcp, there should be an increased amount of transcription. It is understood that the activation of this fused promoter is induced by the NO produced in the degradation pathway of RDX. Therefore, when this naturally occurs inside the E. coli cell the promoter is induced by the presence of RDX. (Lifshitz et al., 2021) |
| BBa_C0061 | CDS | luxI | The luxI gene is known as an autoinducer synthase for AHL. It synthesizes 3-oxohexanoyl-homoserine lactone, which allows it to bind to LuxR. This enzyme is known for creating acyl-homoserine lactones from normal cell metabolites. |
| BBa_C0062 | CDS | luxR repressor/activator | Once in a complex with HSL, LuxR protein binds to the Lux promoter. This process activates the transcription from the luxR & HSL regulated promoter known as luxpR and represses the transcription of luxpL. |
| BBa_K3257071 | Tag | LVA-ssra degradation tag | This degradation tag usually consists of 11 amino acid residues and LVA stands for the last 3 amino acid residues of the tag. When these sequences are fused after a protein, it will induce for protein degradation to take place. The degradation tag will effectively decrease the protein half-life or the typical length of time that a protein, once translated, will exist in the cell. |
| BBa_J176005 | Protein Domain | mCherry | In the presence of RDX, the reporter gene emission of the red fluorescence 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. |
Supplementary device 1: AHL and LuxR protein generator
As a consequence of not finding all of the homologous regulators of AlgD promoter on E. coli, the team decided to design another device that would successfully generate AHL. This supplementary device initiates its transcription with pLac- Luxl promoter when it is induced by Isopropyl ß-D-1-thiogalactopyranoside (IPTG). This will provoke the expression of the Luxl gene and will lead to the conversion of S-adenosylmethionine (SAM) into acyl-homoserine lactone (AHL). These molecules create signals that are recognized by LuxR-type receptors. Next to it there is a RFP protein generator which serves as a marker to certify the transcription of the first part of the device. Finally, the LuxR is regulated by the PTet promoter, so it can constitutively be expressed.
Figure 2. SBOL representation of Supplementary Device 1 genetic circuit.
Table 2. Parts of Supplementary Device 1
| BioBrick | Type | Part Name | Function and Usage |
|---|---|---|---|
| BBa_K876014 | Composite | PLac-LuxI Composite | Transcription of the pLac-LuxI constitutive promoter is induced by IPTG (Isopropyl ß-D-1-thiogalactopyranoside), which will instigate the expression of the LuxI gene. LuxI is a synthase that converts S-adenosylmethionine (SAM) into acyl-homoserine lactone (AHL), which is able to diffuse across cell membranes. AHL signal molecules are recognized by LuxR-type receptors, which constitute a class of transcription factors that possess an amino-terminal AHL binding domain and a carbon-terminal DNA binding domain (Li and Nair, 2012). |
| BBa_K081014 | Generator | RFP Protein Generator | The red fluorescence protein (RFP) protein generator serves as a biological marker to confirm the transcription of the first part of the device. |
| BBa_K116640 | Generator | pTet + RBS + LuxR + T | The LuxR regulated by the pTet promoter is used to constitutively express the LuxR protein. The complex formed by the LuxR activator protein and autoinducer AHL will trigger a transcription response in Device 2 of the right-hand lux promoter, luxpr. |
Device 2: Biodegradation circuit
Starting off the parts of our second device the promoter used was luxpr. It is an inducible promoter activated by the complex formed by the LuxR activator protein and acyl-homoserine lactone (AHL). This activation will allow gene expression of the xplB and xplA genes. The xplB gene codes for a partner flavodoxin reductase and xplA encodes for a flavodoxin domain fused (at the N-terminus) of a P450 cytochrome. XplB and xplA will form a complex that will handle the degradation of RDX such that xplA will catalyze the reductive denitration of RDX and carry out ring cleavage under aerobic or anaerobic conditions reinforced by the xplB gene. This bioremediation of RDX will form two byproducts that will activate our third device: nitrite and formaldehyde. Lastly, the gene amilGFP, with a wavelength spectrum range from 300 – 700nm, is a chromoprotein which exhibits a strong yellow color that will function as our reporter gene.
Figure 3. SBOL representation of Device 2 genetic circuit.
Table 3. Parts of Device 2
| BioBrick | Type | Part Name | Function and Usage |
|---|---|---|---|
| BBa_R0062 | Promoter | Promoter (luxR & HSL regulated -- lux pR) | Promoter inducible by complex formed by LuxR activator protein and autoinducer acyl-homoserine lactone (AHL). AHL induces gene expression by activating the luxpR promoter. This promoter is key for the production of xplB and xplA. The addition of this promoter contributes to our efforts of quorum-sensing and allows a regulated high transcription rate. |
| 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 (Sabir, Grosjean and Bruce, 2017). This allows xplA to catalyze the reductive denitration of 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% (Chong et al., 2014). |
| 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 (Chong et al. 2014), 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 | CDS | 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_B0010 | Terminator | rrnB T1 terminator | The terminator is a sequence inserted to halt the transcription of our first device, based on the BioBrick terminator. |
Device 3: Killswitch Circuit
Our third device is an AND gate that will function as a killswitch and is activated in the presence of formaldehyde and nitrite. It consists of two promoters PyeaR and pfirm. PyeaR is an inducible promoter activated in the presence of nitrite, nitrate or nitric oxide that works effectively under aerobic or anaerobic conditions. Pfirm is also an inducible promoter but is activated by the presence of formaldehyde. Consequently, these promoters are followed by SupD and t7ptag that will function simultaneously in order to activate our T7 promoter. T7ptag is a coding sequence that encodes for a T7 polymerase with two amber mutations that can cancel its translation, but SupD is a tRNA amber mutation suppressor that will eliminate those mutations in t7ptag. Once t7ptag activates the T7 promoter, the production of colicin commences causing bacterial lysis which activates the endonuclease activity in colicin. This then stops the detection and biodegradation of RDX by killing our bacteria and allowing biosafety.
Figure 4. SBOL representation of Device 3 genetic circuit
Table 4. Parts of Device 3
| 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 (T7polymerase 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. |
References
Chong, C. S., Sabir, D. K., Lorenz, A., Bontemps, C., Andeer, P., Stahl, D. A., Strand, S. E., Rylott, E. L., & Bruce, N. C. (2014). Analysis of the xplAB-containing gene cluster involved in the bacterial degradation of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine. Applied and environmental microbiology, 80(21), 6601–6610. https://doi.org/10.1128/AEM.01818-14
Li, Z., & Nair, S. K. (2012). Quorum sensing: how bacteria can coordinate activity and synchronize their response to external signals?. Protein science : a publication of the Protein Society, 21(10), 1403–1417. https://doi.org/10.1002/pro.2132
Lifshitz, A., Shemer, B., Hazan, C., Shpigel, E., & Belkin, S. (2021). A bacterial bioreporter for the detection of 1,3,5-trinitro-1,3,5-triazinane (RDX). Analytical and Bioanalytical Chemistry, (18), 5329–5336. https://doi.org/10.1007/s00216-021-03685-x
Sabir, D. K., Grosjean, N., Rylott, E. L., & Bruce, N. C. (2017). Investigating differences in the ability of XplA/B-containing bacteria to degrade the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). FEMS microbiology letters, 364(14), 10.1093/femsle/fnx144. https://doi.org/10.1093/femsle/fnx144
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