Overview
Cattlelyst is built on three pillars: ammonia conversion, methane oxidation, and safety. Each pillar encompasses several wetlab projects. On this page, we present and highlight the design and results of our work in the lab. Detailed reports on the separate wetlab projects can be found on the Wetlab page or via hyperlinks within this page.
The first pillar of Cattlelyst is based on the conversion of ammonia into dinitrogen gas. We found bacteria able to convert ammonia to dinitrogen gas, in a process called heterotrophic nitrification-aerobic denitrification (HNAD). However, these will most likely not survive in our biofilter, because their natural conditions differ a lot from ours. Being synthetic biologists, we decided to design our own bacterium that can consume ammonia and produce dinitrogen. Before we entered the lab, we developed a dynamic metabolic model to increase our conceptual understanding of HNAD. This process is divided in two different processes, namely nitrification and denitrification. During nitrification, ammonia (NH3) is converted into nitrate (NO3-), nitrite (NO2-) and/or nitric oxide (NO) [1]. Denitrification is the reduction of NO3- or NO2- to the gaseous nitrogen compounds nitric oxide (NO), nitrous oxide (N2O) and N2 [2], [3]. N2O is one of the intermediates in the denitrification pathway and has a global warming potential 300 times that of carbon dioxide over 100 years (Figure 1)[4]. We applied this knowledge in the design of our lab experiments by establishing two important goals: 1) engineering the entire nitrification and denitrification pathways in one organism and 2) limiting N2O production. As our chassis, we chose Pseudomonas putida (P. putida), which is a non-denitrifying, non-pathogenic model organism [5]. For more information, see the engineering cycle page.
Denitrification
Because the exact mechanism and product of nitrification are ambiguous, we decided to introduce the complete denitrification pathway in P. putida. It consists of four enzymes: nitrate reductase (Nap), nitrite reductase (Nir), nitric oxide reductase (Nor), and nitrous oxide reductase (Nos). We developed two approaches to reach this goal: the mosaic approach, and the plug-and-play approach.
Mosaic approach
This approach is similar to the nitrification approach: different genes were combined on a plasmid into one operon with synthetic RBSs between each gene. This way, the enzymes could be separately engineered and tested. Then, active enzymes could be combined in one strain. We aimed to optimize the denitrification pathway and its regulation in a way that NO3- or any other intermediate is funneled through the pathway, avoiding accumulation of (other) intermediates, This should maximize the conversion rate of NH3 to N2.
Plug-and-play approach
Besides the mosaic approach, we created a bacterial artificial chromosome (BAC), carrying all four denitrification operons from P. stutzeri. To guarantee complete transcription of the 31 kb cargo, a T7 promoter was added in the middle, between the Nir and Nor operons. Moreover, at the 5’ end of each operon an RBS was added. High copy number plasmids are normally employed to continuously propagate the system. However, high copy number plasmids containing a large cargo can excessively burden their host and are prone to mutate [13]. Therefore, we integrated the complete cargo in the genome of P. putida EM42 ∆nasT.
To transport the complete denitrification machinery, we prepared a ‘landing pad’ in P. putida EM42 ∆nasT. The landing pad comprises a T7 promoter that controls the expression of Cre recombinase, flanked by two lox sites. The denitrification machinery and gentamycin resistance cassette on the BAC were flanked by compatible lox sites as well. After successful conjugation of the BAC into P. putida EM42 ∆nasT, transiently expressed Cre recombinase recognized the lox sites and subsequently integrated the denitrification machinery.
To test whether the NO2- reductase, and nitric oxide reductase work, we performed a gas chromatography-mass spectrometry (GC-MS) experiment during which we measured the N2O in the headspace. The N2O in normal air is about 8 ppm. We discovered that after 139 hours no N2O accumulates for :SD without IPTG. However, for two out of three biological replicates for :SD + IPTG we did see a slight increase in N2O to 11 and 14 ppm. These findings hint that Nap – Nir – Nor work for P. putida :SD + IPTG.
Denitrification: limiting N2O accumulation
For both the mosaic and plug-and-play approach, we designed an additional project based on CRISPR interference (CRISPRi). With CRISPRi we aimed to redirect the electron flux towards the denitrification machinery.
We learned that the distinct denitrification steps influence each other through electron competition. Pan et al. [15] have shown that the competition arose when the supply of electrons did not meet the demand for electrons by the four reductive denitrification steps. During electron-limiting conditions, electrons are allocated differently to the enzymes, consequently leading to N2O accumulation [15].
P. putida normally uses oxygen respiration as its only electron sink. However, in P. putida :SD, we added the denitrification pathway which is an additional electron sink. By having two electron sinks, we increase the risk that there are insufficient electrons left to fuel N2O reduction to N2, leading to N2O accumulation. Aerobic respiration in P. putida depends on five terminal oxidases, catalyzing the four-step reduction of oxygen to water. To impair P. putida’s ability to dissipate electrons through oxygen respiration, we developed 15 different CRISPRi plasmids targeting the region between the promoter and start codon of these complexes. Given that these genes are essential, we tested for all 15 plasmids if growth was affected (Figure 5). Spacer 4, targeting the promoter of Cyo oxidase, impaired growth during the exponential phase.
The second pillar of Cattlelyst is based on the consumption of methane gas (Figure 6). We discussed why natural methanotrophs such as Methyloccocus capsulatus are not an option in the Engineering cycle. As such, we decided to build a synthetic methanotroph in E. coli, However, before entering the lab we made sure to computationally verify the methane conversion pathway in E. coli using the iGEM PIPE. We used this knowledge to design the pathway for synthetic methanotrophy in C1 consuming strains, that either grown on methane derived products, methanol, formaldehyde, or formate. Both literature and the iGEM PIPE suggested using a methane monooxygenase (MMO) of which two variants exist: the particulate and soluble MMO. The difference between these variants is extensively discussed in the MMO Wetlab page and Engineering cycle page. For our project we followed a multifaceted approach, working to express both variants in E. Coli strains that can use C1 compounds. This would result in a synthetic methanotroph able to consume methane and turn it into biomass or carbon neutral carbon dioxide, an important aspect within the Cattlelyst biofilter.
C1 growing strains
The conversion of methane to methanol does not complete a synthetic methanotroph: methanol has to be converted into either biomass or carbon dioxide. Here two strains that convert methanol or formaldehyde are used. The first strain, C1Saux, is auxotrophic for formaldehyde if grown on minimal medium as it requires formaldehyde or formateto make glycine via the reductive glycine pathway. Additionally, this strain requires the enzyme methanol dehydrogenase (Mdh) to convert methanol into formaldehyde, which has been expressed as shown in [16]. After transformation of this C1Saux strain with the Mdh plasmid, it was able to grow on minimal medium with methanol, as shown in the wetlab MMO page. The second strain is the SM1 strain, which exhibits the Ribulose monophospate pathway and as such can grow with methanol as sole carbon source. Adding a MMO that works in vivo to these strains would allow them to grow on methane. Unfortunately, due to time restraints there was no opportunity to test this.
Methane Monooxygenase
Because we designed our biofilter Cattlelyst for a real-world application on cattle farms, biocontainment of the GMOs is crucial. Therefore, we designed three layers of safety. The first layer is based on the higher methane concentration inside the biofilter than outside of it. This concentration difference is coupled to a methane-dependent kill switch. Another characteristic of the biofilter is the high cell density inside, so the second layer of safety is a proximity kill switch, which is linked to the methane-based kill switch. Thirdly, a co-dependency is added, which makes E. coli and P. putida dependent on each other. As a result, they would starve if they escape the biofilter by themselves. The combination of these three layers of safety ensures total dependency of the two bacterial species on the biofilter’s conditions and on each other, making them unable to escape from the biofilter.
Methane-dependent kill switch
The first layer of our safety mechanisms creates a conditional kill switch in the synthetic methanotroph E. coli that relies on the methane concentration present, which is higher inside the biofilter than outside. A central intermediate of the methane conversion pathway, formaldehyde (CH2O), is used to base the system on [18], [19]. As formaldehyde is a highly (geno)toxic compound [20], it is rapidly detoxified in wild-type E. coli by the FrmRAB operon, consisting of the detoxification genes frmA and frmB, controlled by the repressor frmR of its corresponding promoter Pfrm [21]. This system provides a sensitive formaldehyde biosensor [22], which is coupled to the Hok-Sok toxin/antitoxin system. The production of the toxin Hok can kill the cell by disrupting its membrane structure. It is regulated by a short-lived antisense RNA, Sok, which prevents translation of the hok mRNA [23].
A genetic circuit is designed that contains the FrmR protein as formaldehyde sensor, which is coupled by means of LacI to the Hok/Sok system. This ensures toxin production and cell death in low methane concentration, and cell survival in high methane concentrations, functioning as a conditional ‘kill switch’ in E. coli. Visit the methane-dependent kill switch page or a more detailed description.
To ensure the biofilter bacteria do not die when the methane concentration in the biofilter temporarily drops, we couple the production of toxin to our second safety mechanism based on cell density (see below) (see our human practices work om how we came to this idea). This is achieved by means of a hybrid promoter, that responds to input signals from both LacI (methane-dependent kill switch) and LuxR (proximity-based kill switch). This way, the bacteria only die when both methane and cell density are low.
This system was designed as a plasmid to be constructed in E. coli, but due to cloning difficulties and time constraints, the complete plasmid was not obtained and its dynamics could not be tested. Additionally, to enable increasing the formaldehyde sensitivity of the system, toxicity tests are performed on several strains containing knock-outs of detoxification genes.
Proximity-dependent kill switch
Co-culture
A co-culture experiment was performed to investigate if P. putida can activate the quorum sensing circuit in E. coli. An AHL producing P. putida strain and a wild type P. putida strain as negative control were spread over agar plates and drops of different dilutions of the reporter E. coli strain were added on top. Pictures of the fluorescence of the plates showed activation of the reporter E. coli strain when grown together with a AHL producing P. putida strain.
Co-dependency
Co-dependency was engineered in addition to the methane dependent kill switch and the proximity kill switch to add another layer of safety to the Cattlelyst biofilter. Co-dependency of the two bacterial species was based on the establishment of a cross-feeding community of Escherichia coli and P. putida reliant on amino acids exchange and carbon-source dependency.
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