Engineering Success

Methodology Overview

1. Isolation of Genomic DNA and Gene Amplification

In the initial phase of this research project, the genomic DNA of P. denitrificans is isolated and extracted. The process begins by collecting bacterial cells and subsequently lysing them to release the genomic DNA. Following DNA extraction using established procedures, specific primers tailored to the gene of interest are designed and synthesized. These primers are then employed in PCR (Polymerase Chain Reaction) to amplify the targeted gene from the isolated genomic DNA. The objective is to generate a sufficient quantity of DNA for subsequent experimental steps.

2. Insertion of Genes into Plasmids and Bacterial Transformation:

Once the gene of interest is successfully amplified, the next stage focuses on its integration into plasmids designed to facilitate expression. These plasmids are carefully constructed to include essential elements, such as promoters and terminators, that regulate gene expression. Importantly, the genes of interest are inserted into the plasmids in a manner that places them adjacent to a Histidine-tag (His-Tag) sequence. This His-Tag is crucial for future purification efforts. Alternatively, the His-Tag sequence can be directly incorporated into the designed primers used during PCR amplification. Following plasmid construction, E. coli BL21 bacterial cells are transformed with these recombinant plasmids. Selection markers, such as antibiotic resistance genes, are employed to identify colonies that have successfully integrated the plasmids.

3. Protein Purification Using His-Tag Purification

With transformed E. coli BL21 cells expressing the desired proteins, the project proceeds to the purification phase. Bacterial cells are cultured under conditions that induce protein expression. Subsequently, the cells are harvested and lysed to release the expressed proteins into the lysate. An essential step in this phase involves employing a His-Tag purification method, often based on affinity chromatography. This method allows for the isolation of the target proteins while eliminating unwanted contaminants. The elution process yields purified proteins that are ready for further analysis and experimentation.

4. Enzymatic Electrosynthesis with Peptide Linkers

In the final phase of the research project, purified proteins, which may include enzymes of interest, are utilized in enzymatic electrosynthesis. This process involves the modification of suitable electrode surfaces, often through the introduction of a linker molecule. Subsequently, the isolated proteins are conjugated to the electrode surface using a peptide linker. Proper orientation and immobilization of the proteins are critical to ensure their enzymatic activity is retained. The modified electrodes are then characterized to evaluate their electrochemical behavior and functionality, paving the way for further investigations and applications.

When designing any engineering project, safety invariably ranks among the foremost priorities. This significance is amplified when delving into synthetic biology, where the creation of biological systems, often involving bacteria, demands an unwavering commitment to safety. On this page, we will iterate the engineering design cycle with safety as our main priority when designing NitraNix. Furthermore, we also reiterate our process in designing our primers as an undergrad team on our own.

Aim

Our project's goal is to develop a biotechnical system that converts aquatic nitrates into gaseous nitrogen, focusing on the denitrification cycle and its essential enzymes: nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase, naturally produced by denitrifying bacteria.

lab picture — **Figure 1.** Íris and Beenish in the lab.

Engineering Cycle: PROJECT DESIGN

Design 1.0: How can we reduce the nitrate concentration through the enzymatic cycle?

The purpose of our design is to reduce nitrate concentration enzymatically. Hence, the first step was to define which enzymatic cycle would be appropriate to be utilized to reduce nitrate concentration in our system.

In the nitrogen cycle there are five different stages: fixation, ammonification, nitrification and denitrification. Two of which could reduce nitrate concentration. A reduction to ammonia however was excluded, because the assimilation of ammonia plays a major role in eutrophication of water bodies in general. Therefore, the denitrification cycle, which produces unproblematic gaseous nitrogen, was chosen.

Build 1.0: Choosing the right enzymes and the corresponding bacteria

Having chosen the enzymatic cycle, the next step involves selecting the enzymes and identifying the suitable bacterial strain for constructing our model or system.

Since denitrification entails a four-step process, it is imperative that we isolate four enzymes. To enhance practicality, it is essential that all these enzymes originate from a single bacterium. Additionally, to streamline our system, we must select bacteria with high oxygen tolerance to circumvent the need for intricate incubation conditions. Aerobic denitrification is performed by various α-, β- and γ-proteobacteria like Pseudomonas stutzeri, Pseudomonas aeruginosa, Pseudomonas denitrificans, Ochrobactrum, Pannonibacter, Gordonia, Stenotrophomonas, Paracoccus denitrificans and Azotobacter vinelandii. For electing the bacteria, various terms like the cultivation temperature, the pH and the needed resources of the medium as well as the incubation time were compared. Especially important was the coverage of the sequenced denitrification enzyme and the respective research of the denitrification cycle in the bacteria species.

Test 1.0: Biosafety levels of bacterial candidates

After selecting several bacterial candidates, it's essential to evaluate the pros and cons associated with each of them based on specific criteria. Pseudomonas stutzeri, Pseudomonas aeruginosa and Paracoccus denitrificans were our main contenders.

representation table — **Figure 2.** Representation of various aerobic denitrifiers with selected data. Potential candidates in green. Selected Bacterium in blue.

Here we considered that Pseudomonas aeruginosa is pathogenic with BSL-2 and Pseudomonas stutzeri an optimistic pathogen BSL-1. After weighing each pros and cons, we settled on Paracoccus denitrificans. The bacterium Paracoccus denitrificans was chosen due to its short incubation time, sequenced and studied denitrification genes, facultative anaerobic condition and in particular the ability to express the perisplasmatic nitrate reductase in aerobic and anaerobic conditions.

Learn 1.0: Conclusions on our initial design

Based on the gathered information, we've formulated the concept of our project—a biotechnical system for water denitrification. We've also learned the importance of avoiding enzymes that convert nitrate to ammonia and selecting non-pathogenic bacteria.

Design and Build 2.0: Designing the bioreactor with chosen enzymes and bacteria

The electrochemical system can be constructed using either a microbiological or enzymatic approach. Working with microbes requires constant life-preserving measurements and can reduce denitrification efficiency due to competition with oxygen as the preferred terminal electron acceptor.

Test 2.0: Consultation and Community Engagement

To comprehensively assess our approach, we sought expert advice from Dr. Nils Cremer, a geologist in water management. Our discussion revealed why biological approaches, like ours, are relatively uncommon. We also explored public perceptions of genetically modified bacteria, even with stringent biosafety measures. Recognizing the potential risks associated with the release of modified bacteria, our primary objective is to develop a cell-free electrosynthesis system for denitrification enzymes.

Learn 2.0: Finalizing project design

Based on the information gathered, we finalized our project design. We intend to isolate four enzymes and utilize them in a cell-free system to prevent unintentional bacterial release. The enzymes' activity will depend on electrical current, ensuring safety for end-users.

Engineering Cycle: PRIMER DESIGN AND PCR PROTOCOL

For setting up electrosynthesis chambers, a high amount of purified proteins of the denitrification enzymes is needed. Therefore, a constituent expression of the denitrification genes is needed. P. denitrificans has regulatory inhibition mechanisms of its gene expression in place. Consequently, we needed to clone the respective genes into another organism without these regulatory controls. For effective transformation and expression, E. coli (BL21) is an ideal strain to clone into, due to its engineered capacity to produce T7 polymerase.

planned workflow scheme — **Figure 3.** Planned workflow of the Cloning of Genes NapA, NirS, NorB and NosZ into **E coli**.

pET-23a (+) plasmid scheme — **Figure 4.** Scheme of pET-23a (+) plasmid.

Design 1.0: Plasmid design for various genes insertion

Our design aims to enhance the expression of selected enzymes by amplifying their corresponding genes. The target vector should encompass essential elements, including an origin of replication, a selection gene (such as antibiotic resistance), and both a promoter and terminator. In consideration of our purification method utilizing Histag purification, we require histidine repeats at the end of the expressed sequence. Consequently, we've opted for the pET-23a(+) plasmid, which conveniently incorporates a built-in His-Tag sequence. Since the His-tag sequence is located just before the XhoI recognition site, we aimed to insert our sequence between the XhoI and NdeI site (just before the ribosomal binding site).

Design 1.1: Primer design using NCBI Blast

For primer design, we employed NCBI Primer-BLAST, an online tool that tailors primers to the PCR template, considering factors like primer length, G-C percentage, and annealing temperature.

Test 0.0: In-SIlico PCR

We conducted in-silico PCR tests on all our designed primers.

Build and Test 1.0: Building and Testing our constructs with SnapGene

Based on successful in-silico PCR results, we tested our primer designs with our construct to assess the feasibility of flanked sequence insertion and to preemptively troubleshoot potential issues.

Learn 1.0: Conclusions on our initial designs

Our in-silico PCR and gel electrophoresis experiments revealed errors in our designs. We identified that the XhoI recognition site was present in two of the four genes (norB and nirS) we aimed to amplify and express. This suggests problems during ligation, potentially causing fragmentation of our flanked sequence.

Design 2.0: Redesigning Constructs for norB and nirS

To eliminate the XhoI cutting site while maintaining the plasmid's size, we opted to insert the sequences between BlpI and NdeI. This modification prevents excessive plasmid enlargement and preserves essential regulatory sequences, such as promoters and antibiotic resistance.

Build 2.0: Incorporating His-Tag Sequences

The revised design omits the built-in His-tag sequences required for future purification. As a solution, we decided to include His-tag sequences directly in the primers.

Test 2.0: Evaluating New Designs with SnapGene

Given the relatively long length of the newly designed primers, we subjected them to testing via in-silico PCR and integrated them into our plasmid backbones.

Learn 2.0: Finalizing our primer designs

Our learning process revealed that despite their length, the designed primers are functional. Armed with insights from our in-silico analysis, we are more confident in advancing our project.

DNA-fragment	length
napA	~2.5
nirS	~1.8
norB	~1.4
nosZ	~2

Table 1. Length of DNA-Fragments

Basic Parts created from this Engineering Cycle Go to basic parts page

napA forward: BBa_K4816001
napA reverse: BBa_K4816002
nirS forward: BBa_K4816003
nirS reverse: BBa_K4816004
norB forward: BBa_K4816005
norB reverse: BBa_K4816008
nosZ forward: BBa_K4816006
nosZ reverse: BBa_K4816007

Gene Documentation

Here, you'll find a document that summarizes our comprehensive literature research and the in-silico analysis of the genes, which represents a significant outcome of our rigorous engineering cycle.