RUM-UPRM Wiki Source Code

Project Design

Overview

  Our Prototype consists of two genetic circuits: one for the bioremediation of Mercury and the second for the biodegradation of RDX. Each consists of three devices, each with distinct purposes that activates a chain reaction resulting in the detection of the contaminant, bioremediation or biodegradation, and lysis of the chassis.

Mercury Genetic Circuit

 The Mercury device is composed of three genetic circuits, each with a specific function: detection, bioremediation, and induction of self-lysis to assure biocontrol, which will be regulated by quorum sensing. Upon detection of Mercury, in the first circuit, PmerT will initiate transcription of essential mercury transporter proteins such as MerP and MerT, which will enter the intracellular space. Next, the genes LuxI and LuxR will produce a synthase that will convert normal cell metabolites to acyl-homoserine lactones (AHL) that will bind to the LuxR protein, acting as a transcription factor. The binding of these two molecules to the LuxpR promoter will begin the transcription of the second device, that will contain the MerA and MerB genes, necessary for the bioremediation of mercury. MerB protein works as a lyase, cleaving the bond between the carbon and mercury atoms, converting organic mercury into its elemental form. MerA protein works as reductase which transforms Hg(II) into its gaseous state, which is less toxic and then luciferase will serve as our reporter gene. Finally, the kill switch in the third device activates in the presence of blue light and the binding of EL222 protein, therefore, initiating the transcription of a lysis gene.

  Figure 1: SBOL system for the Mercury Genetic Circuit.

Device #1: Detection and Absorption of Mercury

 In the presence of Mercury, PmerT will initiate the transcription of the bioremediation proteins. Once this promoter is activated, LuxI synthase proceeds to convert S-adenosil metionina (SAM) into acyl-homoserine lactone (AHL) and will also initiate the transcription of the LuxR gene. Then, the transmembrane protein MerP combined with MerT, a periplasmic transporter, will be responsible for transporting Hg(II) into the cytoplasm of the bacterial cell.

  Figure 2: SBOL representation of the Mercury Device #1 Detection.

Table 1: Parts of the Mercury Device #1 Detection.

Part	Function
PmerT BBa_K346002	A mercury-responsive promoter.
merT BBa_K1420005	A transmembrane protein that assists in transporting Hg(II) into the cytoplasm of the bacterial cell. It accomplishes the transport of Hg(II) by interactions of cysteine residues along the protein and folding into the lowest possible energy structure.
merP BBa_K1420003	Gene that encodes for the enzyme MerP which is a periplasmic transporter that brings mercury into the cytoplasm of the cell.
LuxI BBa_C0061	A synthase that converts S-adenosil metionina (SAM) into acyl-homoserine lactone (AHL), which is a small molecule that can diffuse across cell membranes.
LuxR BBa_C0062	LuxR produces a protein that can bind to AHL, stimulating transcription from the right hand lux promoter (pLuxR).

Device #2: Bioremediation of Mercury

Once the LuxR activator protein forms a complex with AHL, pLuxR will initiate the transcription of the proteins downstream that will then increase the rate of transcription and mediate the final effects of quorum sensing. Once the pLuxR promoter is activated, merB catalyzes the breaking of bonds between the organic radicals and mercury, releasing Hg(II). Mercuric ion reductase, MerA, then proceeds to reduce Hg(II) to Hg(0). When the bacteria senses blue light, EL222 is activated, causing the LOV-HTH interaction to occur, which dimerizes the protein and binds its DNA region. LuxAB binds capraldehyde and produces luminescence, indicating that the bioremediation process was successful.

Figure 3: SBOL representation of the Mercury Device #2 Bioremediation.

 Table 2: Parts of the Mercury Device #2 Bioremediation.

Part	Function
LuxR promoter BBa_R0062	The lux pr promoter will be up-regulated by the activation of LuxR protein which forms a complex with AHL, the auto-inducer. This promoter is the key element to produce the proteins of interest, increase the rate of transcription, and mediate the final effects of quorum-sensing.
merA BBa_K1420002	MerA is a mercuric ion reductase that is responsible for reducing mercury from Hg(II) to Hg(0), its volatile and less toxic form.
merB BBa_K1420002	An organomercurial lyase that cleaves the binding between organic radicals and mercury, releasing Hg(II).
EL222 BBa_K2332004	The EL222 gene produces a photosensitive DNA-binding protein that is naturally produced from the marine bacterium Erythrobacter litoralis HTCC2594. EL222 can be found as its inactive or active form. Blue light (450nm) is a factor that can induce the activation of the protein. This causes LOV-HTH interaction to occur, which lets the protein dimerize and bind its DNA region.
LuxA and LuxB BBa_K2310100	The LuxA and LuxB luciferase is a heterodimeric enzyme composed of α and β-subunits where the α subunit is responsible for the light-emitting reaction and the β-subunit stabilizes the protein. It is commonly known as a luciferase and is produced in luminous bacteria where it catalyzes bioluminescence reactions. Lux A and B can act as a reporter system when paired with capraldehyde since it produces luminescence.

Device #3: Killswitch Mercury Parts

This device regulates the population of bacteria in the enclosed system. The Pblind promoter will be activated by using blue-light induction and production of EL222 protein from device #2. This protein is crucial in the activation process of Pblind, an inducible promoter that allows RNAP to transcribe the adjacent lysis gene, which produces colicin, ultimately causing bacterial lysis.

Figure 4: SBOL representation of the Mercury Device #3 Killswitch.

Table 3: Parts of the Mercury Device #3 Killswitch.

Part	Function
Pblind BBa_K2332002	An inducible promoter that is activated in the presence of blue light. To effectively enable this promoter, EL222 protein must be available from RNAP recruitment, as it will permit transcription. Strictly, RNAP will transcribe genes downstream due to EL222, which, ultimately was produced by the blue-light induction.
Lysis gene BBa_K117000	This gene encodes for the lysis protein in colicin-producing strains of bacteria that will result in an interruption of the system by lysis of the bacteria.

RDX Genetic Circuit

The RDX system is composed of three genetic devices, each with a specific function: detection, biodegradation, and lysis, which will be regulated by quorum sensing. This device begins with the stress sensitive promoter algD, which will initiate transcription in the presence of RDX. Later, LuxI gene will create a synthase capable of creating acyl-homoserine lactones (AHL) that will bind to LuxR protein. The binding of these two molecules creates a transcription factor that will activate LuxpR promoter. It will then begin the transcription of the second device, that will contain the xplAB gene, which produces enzymes capable of degrading RDX. After transcription of xplAB gene has been completed, the GFP gene will be transcribed. GFP allows us to identify whether xplAB enzymes are being produced by emitting a green fluorescence. The end-products of RDX, specifically nitrite, and formaldehyde, will act as transcription factors in the third device, which is the killswitch. Lastly, the kill switch circuit will be controlled by a modified synthetic-AND Gate, which will allow bacterial lysis by requiring the presence of the byproducts of the biodegradation of RDX: formaldehyde and nitrate. Lysis will initiate due to the presence of colicin and, therefore, stop bacterial transcription.

Figure 5: Overview of the SBOL system for the RDX Genetic Circuit.

Device #1: Detection of RDX

In the presence of RDX, algD promoter will initiate the transcription. As the transcription begins, luxI gene will convert S-adenosil metionina (SAM) into acyl-homoserine lactone (AHL); consequently the luxR will produce a protein which binds to AHL. This merge will stimulate the transcription of luxpr (pLuxR) promoter in the second device.

Figure 6: SBOL representation of the RDX Device #1: Detection.

Table 4: Parts of the RDX Device #1: Detection.

Part	Function
algDpromoter	Transcription of this promoter begins due to a stress response towards RDX.
LuxI BBa_C0061	This synthase converts SAM into a small molecule called an acyl-homoserine lactone (AHL), which can diffuse across cell membranes.
LuxR BBa_C0062	When bound to AHL, it produces a protein that can stimulate transcription from the right hand lux promoter (LuxpR).

Device #2: Biodegradation of RDX

The LuxpR promoter will be upregulated by the activation of LuxR activator protein, which forms a complex with autoinducer AHL. As a result, the xplAB system will catalyze the reductive denitration and subsequent ring cleavage of RDX. When biodegradation of RDX is complete the gene GFP, a green fluorescent protein, will function as a reporter gene.

Figure 7: SBOL representation of the RDX Device #2: Biodegradation.

Table 5: Parts of the RDX Device #2: Biodegradation.

Part	Function
luxpR BBa_R0062	Promoter that will be up-regulated by the activation of LuxR activator protein which forms a complex with autoinducer AHL. This promoter is the key element to produce proteins of interest, increase the rate of transcription, and mediate the final effects of quorum-sensing.
xplA and xplB genes	Involved in the catalyzation of the reductive denitration and ring cleavage biodegradation pathways for the organic contaminant RDX. The xplB gene encodes for a partner flavodoxin reductase, while the xplA encodes for flavodoxin domain fused (at the N-terminus) of a P450 cytochrome.
merB BBa_K1420002	An organomercurial lyase that cleaves the binding between organic radicals and mercury, releasing Hg(II).
GFP with degradation LVA tag BBa_K592010	Involved in the expression of green fluorescence protein, as well, encodes for a small peptide functioning as a degradation tag that will allow for fine-tuning protein levels and thus regulating of the GFP in the bacteria.

Device #3: Killswitch of RDX Circuit

To maximize efficiency of our prototype, we decided to harbor the use of modified synthetic-AND Gate as our killswitch, which was originally developed by Christopher A. Voigt and modified by the Peking University 2009 iGEM team. Our synthetic AND Gate requires the use of two inputs, formaldehyde and nitrite, which are the byproducts of biodegraded RDX, to generate the protein colicin, which causes cellular lysis. The lysis gene (output) will have an inducible T7 promoter which will be activated with the corresponding T7 RNA polymerase. This polymerase (T7ptag) will be added to the circuit and will be regulated by an inducible promoter, PyeaR, which will activate in the presence of nitrite. However, this polymerase will have two amber mutations, which are nonsense mutations that inhibit the complete translation of the polymerase. To overcome the nonsense mutation, a tRNA amber mutation suppressor, SupD, will be controlled by a formaldehyde-inducible promoter, Pfrm. This means that lysis will only occur if both nitrite and formaldehyde are present.

Figure 8: SBOL representation of RDX Device 3: Killswitch.

Table 6: Parts of RDX Device 3: Killswitch.

Part	Function
Pyear promoter BBa_K216005	Inducible promoter that will be activated in the presence of nitrate, nitric oxide, or nitrite.
Formaldehyde-Inducible Promoter BBa_K2728001	Inducible promoter that will be activated in the presence of formaldehyde.
SupD + terminator BBa_K228100	A tRNA coding gene and it can be well terminated by the terminator BBa_B0015.
T7ptag (T7polymerase with amber mutation) BBa_K228000	A coding sequence of T7 polymerase with two Amber mutations. The transcription of T7ptag gene can only lead to the generation of its mRNA, further translation into T7 RNA polymerase is blocked because of the amber mutation.
PT7 BBa_K2406020	When the T7 RNAP is present it permits levels of transcription.
Lysis gene BBa_K117000	This gene encodes for the lysis protein in colicin-producing strains of bacteria that will result in an interruption of the system by lysis of the bacteria.

References

 [1] Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, Molin S. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol. 1998 Jun;64(6):2240-6. doi: 10.1128/AEM.64.6.2240-2246.1998. PMID: 9603842; PMCID: PMC106306.

 [2] Chong CS, Sabir DK, Lorenz A, Bontemps C, Andeer P, Stahl DA, Strand SE, Rylott EL, Bruce NC. 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. 2014 Nov;80(21):6601-10. doi: 10.1128/AEM.01818-14. Epub 2014 Aug 15. PMID: 25128343; PMCID: PMC4249041.

 [3] Halasz, A., Manno, D., Perreault, N. N., Sabbadin, F., Bruce, N. C., & Hawari, J. (2012). Biodegradation of RDX Nitroso Products MNX and TNX by Cytochrome P450 XplA. Environmental Science & Technology, 46(13), 7245-7251. doi:10.1021/es3011964

 [4] Jackson, R. G., Rylott, E. L., Fournier, D., Hawari, J., & Bruce, N. C. (2007). Exploring the biochemical properties and remediation applications of the unusual explosive-degrading P450 system XplA/B. Proceedings of the National Academy of Sciences, 104(43), 16822-16827. doi:10.1073/pnas.0705110104

 [5] Lee, B., Baek, H., & Oh, K. (2013). Use of an algD Promoter-Driven Expression System for the Degradation of Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Pseudomonas sp. HK-6. Current Microbiology, 67(4), 480-486. doi:10.1007/s00284-013-0387-5

Team:RUM-UPRM/Design