Team:Lambert GA/Nar

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NAR

PURPOSE

Nitrate is an important nutrient for plants in hydroponics systems. To develop a user-friendly hydroponics system, we extensively researched and engineered a biosensor to detect nitrate. Traditionally, electrical sensors can be expensive, and basic paper test strips lack the sensitivity needed to operate a small-scale hydroponics system. The 2020 team designed both nitrate and nitrite biosensors based on the native Nar operon in E. coli, which is responsible for controlling nitrogen-based reactions in the cell. As a continuation of our 2020 project, we implemented our previous nitrate cell-based biosensor into a cell-free system to address biosafety issues and the iGEM “Do Not Release” policy regarding the distribution of cells to the general public.

NARL SIGNALING PATHWAY | NAR OPERON

See Lambert_GA 2020: Nar Operon

Figure 1. Diagram of the E. coli native Nar membrane-bound sensor proteins and their corresponding DNA-binding response regulators. In vivo, nitrate can also phosphorylate NarQ however it is insignificant.

The native E. coli 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 their conjugate DNA-binding response regulators, NarL and NarP (figure 1). Lambert iGEM’s system utilizes the membrane-bound sensor protein NarX and its complement NarL. NarX senses the amount of nitrate present. In the presence of nitrate, NarX will phosphorylate NarL activating it[2]. Conversely, if no nitrate is present, NarX will neither phosphorylate nor activate NarL. The Phosphorylated NarL-Specific Promoter Sequence (PNar) triggers the expression of sfGFP in the presence of phosphorylated NarL[2], thereby allowing for the quantification of nitrate (See Lambert_GA 2020: Nar). Utilizing this pathway our team created an ordinary differential equation (ODE) model to mathematically correlate the GFP expression to nitrate through fluorescence data [1].

PROCESS

Cell-free systems allow for cellular processes to occur in vitro, eliminating both the physical constraints of a living cell and the safety hazard of live cell distribution [3]. This is achieved through the preparation of a lysate that removes genomic DNA and the cell membrane, resulting in a solution with isolated cell parts later used for synthesis.

The base of a cell-free system is the lysate, which consists of isolated cell parts later used for protein synthesis. A preculture of cells was cultivated in LB for 16 hours. Then, 20mL of this preculture was added to 480mL of 2X YT+P growth media and was grown to OD600 1.0. The cells were then centrifuged and washed to achieve a cell pellet, which was lysed by sonication. The lysis product was centrifuged to separate lysate from debris, and a run-off reaction was performed. This product was dialyzed and finally centrifuged once again to isolate the final plasmid, which was aliquoted and stored at -80ºC (figure 2).

Figure 2. Aliquoted plain BL21 cell lysates.

In addition to the lysate, a cell-free system needs purified plasmids and a master mix containing an energy source, salts, and amino acids to run. We purified our plasmids with the Omega BioTek Miniprep Kit to eliminate residual RNAse contamination that could affect experimental results. The master mix was prepared using five different solutions: salt stock, NTP master mix solution, energy stock, reagent stock, and amino acid stock. These solutions were prepared separately then combined to create a final master mix.

OBSTACLES

OBSTACLES | MEMBRANE-BOUND PROTEIN

The nitrate (NarL/NarX) biosensor relies on a membrane-bound sensor protein NarX to activate a DNA-binding response regulator. Cell-free lysates with membrane-bound proteins, or enriched lysates, are relatively new and difficult to work with.

To formulate an enriched lysate with NarX protein embedded in the membrane mimicking lipids, we have to prepare lysates from cells expressing NarX. Utilizing plasmid pBL124-4, designed and provided by Dr. Adam Silverman (See Lambert_GA 2021: Integrated Human Practices), we cultured cells that over-express NarX, leading to slower growth. In order to have active NarX protein in the cell membrane, careful sonication was necessary to ensure that we lyse our cells without impairing the NarX protein.

TROUBLESHOOTING

Because our team is new to the cell-free system, we encountered difficulties with lysate preparation. In our high school lab setting, we had limited access to equipment.

1. Our protocol recommended spinning the cells at 4000xg while washing at the beginning of lysate preparation (See protocol for more details), but the centrifuge we had access to had a maximum speed of 3260xg. This caused initial problems with pelleting because the centrifugal force was not strong enough. To account for this, we decrease our extraction size from the suggested 1L to 500 mL and centrifuged for 15 minutes rather than 10.
2. With limited settings on our sonicator (figure 3), we could not regulate the voltage applied to lyse our cells. The lowest sonication setting on our device was higher than the recommended amount, so we resorted to visual cues as an endpoint signal (figure 4). This lead to large variability in lysate clarity.

Figure 3. Sonication set up in our lab

Figure 4. Visual difference between unsonicated (left) and sonicated (right) cells. The murky cell resuspension turns more translucent after sonication.

EXPERIMENTATION

After we successfully prepared a lysate from BL21(DE3) competent E.coli cells and another NarX-enriched lysate, we ran two experimental procedures. We used an experimental spreadsheet provided by Megan McSweeny (figure 5) to calculate the amount of plasmids to add to our lysates. The final volume of our reactions were 34µL, which was then allotted into 10µL for each well on the well plate to have technical triplicates.

Figure 5. Spreadsheet provided by Megan McSweeny from StyLab at Georgia Institute of Technology to aid our experimental calculations.

1. To determine if all the solutions in our master mix are working, we first experimented with BL21(DE3) competent E.coli cells and three Lac-Operon-based plasmids provided by Dr. Silverman (figure 6).
   

   Figure 6. Chart for cell-free lysate experimentation using LacI, LacO/GFP, GFP, and IPTG.

2. After our initial experimentation yielded successful results, we began testing our enriched lysate with nitrate concentration between 0 and 300 ppm in conjunction with the NarX, NarL, and pNar plasmids (figure 7), again provided by Dr. Silverman.
   
   
   

   Figure 7. Chart for cell-free nitrate sensor experimentation using NarX, NarL, pNar/sfGFP plasmids, IPTG, and varied amounts of nitrate concentrations.

RESULTS

Our initial set of Lac-Operon tests showed us our lysate is working. Our lysate produced GFP when plasmid 343, a T7 promoter driving expression of GFP, was added to our lysate. Inducing the GFP with IPTG showed a significant increase in fluorescence.

Figure 8. Plate Reader fluorescence data for T7 GFP and T7 GFP + IPTG.

After our initial experimentation yielded successful results, we tested the same lysate with our Fusarium and Phytophthora plant pathogen toehold and trigger plasmids (See Figure 8). Preliminary tests did not reveal a statistical difference between the baseline and in the presence of plasmids. We are continuing to troubleshoot our experimentation protocols to successfully implement both plant pathogens into a cell-free system.

In addition, we have begun testing the efficacy of our NarX enriched lysates. We plan to test our lysates with varying concentrations of NarX, NarL, and pNar plasmids and nitrate concentrations ranging from 0 ppm to 300 ppm (See Figure 7).

After our initial experimentation yielded successful results, we tested the same lysate with our Fusarium and Phytophthora plant pathogen toehold and trigger plasmids (See Figure 9). Preliminary tests did not reveal a statistical difference between the baseline and in the presence of plasmids. We are continuing to troubleshoot our experimentation protocols to successfully implement both plant pathogens into a cell-free system.

Figure 9. Cell-free experimental set up for the plant pathogen biosensors.

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] 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

[3] Silverman, A. D., Kelley-Loughnane, N., Lucks, J. B., & Jewett, M. C. (2018). Deconstructing Cell-Free Extract Preparation for in Vitro Activation of Transcriptional Genetic Circuitry. https://doi.org/10.1021/acssynbio.8b00430.s001