Design
Objective
To effectively implement diazotrophic nitrogen fixation in E.coli using the Nitrogenase pathway and ensure its optimal operation under varying oxygen conditions.
The Mechanism
- Diazotrophs: Specialised bacteria capable of converting atmospheric nitrogen (N2) to bioavailable ammonia (NH3).
- Nitrogenase Pathway: Employed predominantly by diazotrophs, this pathway revolves around two core structural proteins - Reductase (sourced from nifH) and Nitrogenase (from nifD & nifK). To facilitate the pathway, additional regulatory genes and proteins are essential, with nifA playing a regulatory role in overseeing nitrogenase protein synthesis.
Challenges and Solutions
Oxygen Sensitivity: When interacting with oxygen, nitrogenase can generate reactive oxygen species (ROS) which can be harmful to the cell, thus, ensuring reasonable efficiency mandates careful regulation in response to oxygen concentrations, with levels above 2% O2 considered detrimental. Our solution thus lies in harnessing an inherent oxygen-sensitive system in e.coli, fumarate and nitrate reductase (FNR), to upregulate transcription in optimal conditions while deactivating in high oxygen, reducing transcription thus ensuring cell safety.
Test Structure
Our designs tested 3 promoters, narGp, narKp and dmsAp each modified to possess an extra half binding site [TTGAT] (1.5 total binding sites each) with the intention of increasing FNR sensitivity to maximise yield in low O_2 conditions.
With the final intention to have FNR upregulate nifA, our test structure utilised green fluorescent protein (GFP) as a placeholder for nifA while varying the promoter interacting with FNR to optimise yield. In addition to differentiating via the intensity of fluorescence (indicating rate of transcription), different oxygen levels were also tested ensuring transcription is adequately oxygen-sensitive and is sufficiently reduced with >2% O_2 concentration.
Part Design
Our designs tested 3 promoters, narGp, narKp and dmsAp each modified to possess an extra half binding site [TTGAT] (1.5 total binding sites) with the intention of increasing FNR sensitivity to maximise yield in low O_2 conditions.
Our oxygen prototype consisted of the following 3 parts (parts 2 and 3 were put together in the registry):
- Part 1 constant (BBa_K4735012): lacIQ promoter which provides baseline rate of transcription for rbs-fnr followed by a TrrnBT1 terminator. This provides a constant supply of FNR protein
- Part 2 variable (BBa_K4735013, BBa_K4735014, BBa_K4735015 (all include part 3)): A T-Spacer and T7 binding site (for future positive control testing), followed by the promoter to which FNR will bind, each containing 1.5 binding sites, varies between a narGp, narKp and dmsAp promoter.
- Part 3 constant: Ribosome binding site (different to the rbs in part 1) rbs2 followed by superfolder fgfp and then a terminator TrrnBT1.
Primers for PCR amplification + Golden Gate Assembly
For assembly, we utilised the NEBridge golden gate assembly tool to design primers for PCR, yielding:
- Prototype A = Part 1(ac) + Part 2a(a) + Part 3(ac)
- Prototype B = Part 1(b) + Part 2b(b) + Part 3(b)
- Prototype C = Part 1(ac) + Part 2c(c) + Part 3(ac)
Where: Prototype n = Part 1(primer) + Part 2n(primer) + Part 3(primer)
Build
Delivery of parts and amplification via PCR
After performing a quality check confirming the quality of delivered parts with gel electrophoresis, PCR protocol was followed to amplify parts 1, 2, and 3 for prototypes A, B, and C with primers indicated above, after which another gel electrophoresis was performed confirming parts were correctly amplified.
Golden Gate Assembly
We mix the our parts together in the molar ratio described in the kit, however, the provided vector was not used, and instead a 1:1:1:1 ratio of Part 1, Part 2, Part 3, and pSB1C3 (amplified BioBrick Vector with BsaI sites) was used.
We followed golden gate assembly protocol to assemble our plasmid as per the ratio above and incorporated this into our transformant NEB 5-alpha competent E.coli cells.
As a final quality check, we screened transformants and extracted plasmid DNA, which was tested via gel electrophoresis and 3rd party sequence verification.
Test
Qualitative Observations
Utilising handheld UV testing on agar plates, the viability of the GFP in prototype cells was confirmed by observing fluorescence.
Qualitative Experiments
Our e.coli was incubated into liquid culture set to desired atmospheric oxygen levels of 0% (anaerobic), 0.5%, 2%, and aerobic conditions (~18%). Once cells were given time to incubate bacteria growth was measured using a microplate reader, then the fluorescence intensity of GFP at each condition was recorded. Analysis was then performed adjusting for relative intensity yielding a graph, normalised to OD600, portraying the relative fluorescence of each promoter compared to a control with no GFP (A, B, and C is NarG, NarK, and DmsA respectively).
Learn
Our experiment shows that our test cycle succeeded as FNR sensitivity of oxygen was shown to be successful as an oxygen sensor with promoters B (NarK) and C (DmsA) whereas it showed no O2 sensitivity in promoter A (NarG). As shown on the graph, high fluorescence intensity was observed when our prototypes were incubated in oxygen conditions below 2%. For the purposes of our project as a whole, we’ve determined that promoter B of NarK is most suitable for the continuation of our project as it exhibited more stable, consistent fluorescence across different oxygen intervals with a more compact spread of results than promoter C (DmsA) between the biological replicates.
As seen in the graph above, there is an increase in GFP production in both successful promoters from 0% to 0.5%. Also, both successful prototypes became inactive at ~18% oxygen approaching atmospheric conditions as expected. Hence, both systems successfully function as an oxygen sensor to the requirements of our experiment.
Future Endeavours
Following the success of FNR oxygen sensitivity, our next step is to substitute the sfGFP indicator with NifA, which regulates nitrogenase production. We believe that this will maximise the efficiency of nitrogenase production, by optimising its production to only occur when nitrogenase can function, under the ideal oxygen concentration levels. Through maximising the production of nitrogenase, we should be able to efficiently produce ammonia within the soil, effectively revitalising the ammonia content of the soil in a self-sustaining manner.
After incorporating the NifA regulator, future efforts could focus on developing a sensor to other soil factors, such as carbon-source availability, and concentration of minerals such as Iron and Molybdenum that are required in the process of nitrogen fixation. By successfully optimising our E.coli to be able to sense a multitude of environmental conditions, the efficiency of nitrogen fixation can be greatly enhanced.
Citations
Ryu et al., (2020). Control of nitrogen fixation in bacteria. Nature Microbiology.
Kiley, P. J., & Beinert, H. (1998). FNR's role in oxygen sensing. FEMS Microbiology Reviews.
Martinez-Argudo, I., et al. (2004). The multifaceted NifL-NifA System.
