Team:Lambert GA/Nar
BACKGROUND
PURPOSE
To develop a user-friendly aquaponics system, Lambert iGEM extensively researched a biosensor to detect essential nutrients, such as nitrogen in the form of nitrate and nitrite, which plants require. The Nar system, an operon native to E. coli, is responsible for controlling nitrate-based reactions within the cell. Fortunately, HKUST-Rice iGEM 2015 also utilized this pathway for nitrate sensing, and their part became the foundation for Lambert iGEM’s part. To further characterize HKUST-Rice’s BBa_K1682018, the team’s lab conducted comprehensive research and planned experiments with the part in the lab, both virtually and in person.
Lambert iGEM’s envisioned Nar system would not only sense nitrate but also nitrite, a novel addition to allow the team to quantify their individual concentrations. Lambert iGEM has constructed a comprehensive BioBrick to detect the presence of nitrite, similarly designed to the aforementioned nitrate-sensing part. Traditionally, electrical sensors for specific nutrients tend to be very expensive, making them inaccessible to the general public. Additionally, paper strips are not exact and lack sensitivity. With Lambert iGEM’s system, the user would receive accurate feedback on the nutrient levels for his/her plants, efficiently sustaining them for longer periods of time and allowing for a higher yield of produce.
NAR OPERON
Figure 1. The diagram above shows the native Nar membrane-bound sensor proteins and the corresponding DNA-binding response regulators in E. coli.
Native to E. coli, the Nar Operon regulates anaerobic gene expression in response to two electron acceptors: nitrate and nitrite. This system consists of two homologous membrane-bound sensor proteins (NarX and NarQ) as well as two homologous DNA-binding response regulators (NarL and NarP). NarL’s conjugate is the membrane-bound NarX protein while NarP’s conjugate is the membrane-bound NarQ protein. Lambert iGEM is utilizing this system to test nitrate and nitrite levels, NarL and NarP expression, and downstream GFP expression through mathematical models [1].
NarL SYSTEM
The NarL system is partially designed after HKUST-Rice 2015’s part BBa_K1682018, modeling after E.coli’s natural nitrate and nitrite sensor. While Lambert iGEM’s NarL system is modified to change the original design’s terminators due to problematic DNA synthesis in IDT (Integrated DNA Technologies), both HKUST-Rice’s and Lambert’s NarL biosensor detect nitrate [2]. The promoter BBa_J23106, an Anderson Promoter, constitutively produces TetR, which represses the PtetO promoter that produces NarL. By regulating the amount of aTc, the molecule that inhibits TetR, the team can control levels of NarL. NarX, the native membrane-bound protein, senses the amount of nitrate in the cell: in the presence of nitrate, NarX will phosphorylate NarL, activating it; however, if there is no nitrate present, NarX will not phosphorylate NarL [1]. The PdcuS promoter, which naturally produces green fluorescent protein (GFP), is repressed by phosphorylated NarL which causes lower GFP levels in the presence of nitrate [3]. Lambert iGEM plans to correlate the GFP levels to nitrate through mathematical modeling.
Figure 2. Diagram of Lambert’s NarL System
NarP SYSTEM
After speaking with Dr. Guerdat, an environmental engineer at the USDA and an expert in aquaponics, the team realized the need for detecting nitrite since certain bacteria (naturally occurring in any environment and not produced by Lambert iGEM) in aquaponics systems generate nitrate from ammonia and the intermediate is nitrite- a toxic molecule to fish. It is imperative that the levels of nitrite are regulated and exact to ensure that the levels are not toxic to any of the system’s components.
Because the NarL system detects nitrate, Lambert iGEM needed a method to detect levels of nitrite: NarP, which detects both nitrate and nitrite. By calculating the corrected difference, the system would have concentrations for both nutrients. Lambert iGEM’s novel NarP composite part BBa_K3411020 and the NarP gene BBa_K3411010 are also modeled after E.coli’s natural nitrite sensor. Similarly to NarL, the promoter BBa_J23106, a constitutive Anderson Promoter, produces TetR, which represses the PtetO promoter, producing NarP. By regulating the amount of aTc, the molecule that inhibits TetR, the lab can control NarP’s expression, the product of the PtetO promoter. NarQ, the native membrane-bound protein, senses the amount of nitrate and/or nitrite in the cell: in the presence of nitrite and/or nitrate, NarQ will phosphorylate NarP, activating it, but if there is no nitrite and/or nitrate present, NarQ will not phosphorylate NarP [1]. If NarP is phosphorylated, it will activate the nirB promoter, producing GFP [4]. Lambert iGEM plans to correlate GFP levels to nitrite through mathematical modeling.
Figure 3. Diagram of Lambert’s NarP System
RESULTS
Lambert iGEM initially attempted to clone its NarL insert into the pSB1C3 plasmid using Restriction Cloning. The team hydrated the NarL insert, digested both the 1C3 backbone (obtained from minipreps) and the insert using EcoRI and PstI, then ligated the insert and backbone. Lambert iGEM then transformed its ligation into E.coli cells. When the plates exhibited no fluorescence rather than GFP, a Colony PCR was run, which indicated that the NarL insert was not taken up and the backbone folded in on itself. To troubleshoot, the team increased ligation’s insert to vector ratio. The subsequent Colony PCR still indicated that NarL was not uptaken. After further research, the team realized that the 1C3 vector was ligating in itself after digestion due to the insert’s large size - 2701 base pairs, making it virtually impossible for the plasmid to uptake the NarL part.
Therefore, Lambert iGEM decided to switch to Gibson Assembly, increasing the probability of insert and backbone alignment. The team successfully amplified the 1C3 backbone; however, when attempting to PCR the insert, Lambert iGEM ran into some challenges. Integrated DNA Technology- the company Lambert iGEM orders DNA from- did not recommend using PCR to amplify an insert larger than 1kb. Therefore, the team would be unable to PCR the Nar Insert, which is approximately 3kb. As a result, Lambert iGEM decided to design primers for the insert to create two separate fragments with an approximate 30 base pair overhang, making each insert closer to the size of the successfully amplified Pho insert (See Wetlab: PHO) . Lambert iGEM is currently testing this method; however, if the team is unable to successfully amplify the two insert fragments, they plan to split the insert into three fragments and use Gibson Assembly to connect the three fragments to the pSB1C3 vector (See Project: Nar Engineering Success) .
FUTURE PLANS
MODELING
Similar to this year’s Pho Regulon signaling pathway model, Lambert iGEM will develop deterministic Ordinary Differential Equation (ODE) models of the Nar Operon next year before characterizing the nitrate/nitrite biosensors in the lab. There will be two models - one for the NarL system, and one for the NarP system. The models will simulate the activity of the biosensors over time and predict specific GFP expression in a single E. coli cell based on initial inputs of extracellular nitrate/nitrite.
The process of building the NarL model will follow this year’s Pho model. Lambert iGEM will diagram all the reactions of the signaling pathway with MATLAB Simbiology software, set initial species values, generate ODE equations, estimate missing rate constants using HKUST-Rice 2015’s characterization data, and simulate GFP expression with varying inputs of nitrate concentrations. For the NarP model, the process will be the same until estimation of rate constants. Since Lambert iGEM will be creating a new part for the NarP system, there is no existing characterization data for NarP; Lambert iGEM will search literature to find missing rate constants and complete the model, then simulate GFP expression.
EXPERIMENTAL
In the coming months, Lambert iGEM plans on conducting trials to characterize NarL’s sensitivity to differing concentrations of nitrate and/or nitrite. By calculating the corrected difference between NarL and NarP, Lambert iGEM can deduce the concentrations of both nutrients since Nar L detects nitrate and NarP detects both nitrate and nitrite.
For further experiments, Lambert iGEM will use HKUST-Rice 2015 Biobrick’s initial characterization of aTc to the resulting NarL levels. HKUST-Rice 2015 found that the 0-20ng/mL aTc range provides the most variation in NarL levels since values above 20ng/mL yield the same amount of NarL as 20ng/mL. Moreover, in an aquaponics system, the fish’s optimal range is no more than 120 ppm while the plants’ optimal range is 100-140ppm, so the team will characterize the NarL biosensor from 0-140ppm. These values were found from Lambert iGEM’s hydroponics’ and fish tank’s water.
Following the Nutrient Mix Committee’s successful results of cells surviving at these specific conditions, Lambert iGEM can test NarL’s sensitivity to nitrate in 0-20ng/mL of aTc. The plan is to create a large batch of the team’s engineered biosensor cells and measure the OD levels so that each tested tube will have an equal amount of engineered cells.
Experimental Testing aTc and Nitrate Concentrations
Table 1. Chart of the various nitrate concentrations (uM) and aTc concentrations that will be used when testing.
Table 2. Chart of the various nitrite concentrations (uM) and aTc concentrations that will be used when testing.
Table 3. Chart of the various nitrite concentrations (uM) and aTc concentrations that will be used when testing.
Lambert iGEM also plans to do the same for NarP, the novel part, while taking into consideration both nitrate and nitrite. Therefore, testing will be done in 3 scenarios: in the presence of nitrate, nitrite, and nitrate & nitrite at differing concentrations as triplicates.
The nitrate and nitrite level for the system on October 26, 2020 was over 250 ppm and 10 ppm respectively. Taking into account that the nitrate concentration was higher than 250 ppm, the team decided to test a range of 0 ppm - 300 ppm for nitrate concentrations. For nitrite, the team decided to set the range as 0 ppm - 20 ppm on an interval of 5, taking into account that the nitrite concentration was measured higher than 10 ppm. However, this did not include the testing for optimal nitrite concentration, which is 0.25 ppm - 1.0 ppm. Thus, Lambert iGEM plans to have another experiment with nitrite level ranging from 0 ppm to 2.5 ppm with 0.5 interval. The reason for high nitrite concentration measurement can be explained by the sudden spike of nitrite level.
KEIO STRAINS
As previously explained, PdcuS is the NarL system’s promoter in that PdcuS is repressed by phosphorylated NarL; however, there is not sufficient research confirming that PdcuS is not also repressed by NarP.
This became an issue since E.coli has both native NarL and NarP. In other words, Lambert’s system would experience background noise, where even if no NarL was present, the PdcuS promoter could still be repressed by native NarP. To combat this issue, the team decided to use a NarP Keio Strain (Strain JW2181-2), eliminating the native NarP’s potential interference in Lambert iGEM’s NarL system.
Likewise, Lambert iGEM’s NarP system uses nirB as its promoter; however, nirB is affected by native NarL, so a NarL Keio Strain (Strain JW1212-1) is similarly used to eliminate the native NarL’s effects on Lambert iGEM’s NarP system.
NarL
Figure 4. On the left depicts an image of the NarL biosensor in a traditional E. coli cell without any knockouts. On the right are the plasmid cells in the NarP Knockout Keio Strain.
Without NarP Keio Strain
As depicted above, even in the presence of no nitrate (what the NarL system is built to measure), NarP will be phosphorylated and could repress the promoter PdcuS, yielding false positives.
With NarP Keio Strain
Only nitrate-activated molecules would repress PdcuS since Native NarL can also only be phosphorylated by NarX (the conjugate membrane-bound protein) in the presence of nitrate.
NarP
Figure 5. On the left depicts an image of the NarP biosensor in a traditional E. coli cell without any knockouts. On the right are the plasmid cells in the NarL Knockout Keio Strain.
Without NarL Keio Strain
As depicted above, even in the absence of nitrite, NarL will be phosphorylated and could activate the promoter nirB, yielding false positives as Lambert’s NarP System is built to measure both nitrate and nitrite. Even though NarL measures nitrate- a goal of the NarP system- it would be to a lesser degree than that of the NarL system’s (built to measure only nitrate) results. This would result in an error in the corrected difference later taken.
With NarL Keio Strain
Only nitrate-activated molecules would repress PdcuS since Native NarL can also only be phosphorylated by NarX (the conjugate membrane-bound protein) in the presence of nitrate.
ACCOUNTABILITY
During the COVID-19 pandemic, Lambert iGEM adapted to work efficiently despite minimal access to the lab or face-to-face interactions. The team decided that the best plan to guarantee efficiency was to do extensive research until the team could return and designed Lambert iGEM’s protocols and parts in advance.
REFERENCES
[1] Darwin A.J., & Stewart V. (1996) The NAR Modulon Systems: Nitrate and Nitrite Regulation of Anaerobic Gene Expression. In: Regulation of Gene Expression in Escherichia coli. Springer, Boston, MA. https://doi.org/10.1007/978-1-4684-8601-8_17
[2] HKUST-Rice iGEM (2015). Potassium, Phosphate and Nitrate Biosensors. Retrieved from https://2015.igem.org/Team:HKUST-Rice/Nitrate_Sensor_PyeaR
[3] Gob E., Bledsoe P., Chen L., Gyaneshwar P., Stewart V., & Igo M. (2005). Hierarchical Control of Anaerobic Gene Expression in Escherichia coli K-12: the Nitrate-Responsive NarX-NarL Regulatory System Represses Synthesis of the Fumarate-Responsive DcuS-DcuR Regulatory System. Journal of Bacteriology, 187(14): 4890–4899. doi: 10.1128/JB.187.14.4890–4899.2005
[4] Wang H., & Gunsalus R. (2000). The nrfA and nirB Nitrite Reductase Operons in Escherichia coli Are Expressed Differently in Response to Nitrate than to Nitrite. Journal of Bacteriology, 182(20): 5813–5822. doi: 10.1128/jb.182.20.5813-5822.2000