iGEM 2023 — Team bonn-rheinbach

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

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* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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## Methodology Overview

![Isolation of Genomic DNA and Gene Amplification](https://static.igem.wiki/teams/4816/wiki/pcr-machine-icon.png)

![Insertion of Genes into Plasmids and Bacterial Transformation](https://static.igem.wiki/teams/4816/wiki/bacteria-with-plasmid-icon.png)

![Protein Purification Using His-Tag Purification](https://static.igem.wiki/teams/4816/wiki/histag-purification-icon.png)

![Enzymatic Electrosynthesis with Peptide Linkers](https://static.igem.wiki/teams/4816/wiki/bioreactor-icon.png)

### 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.

---

## Engineering Success

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](https://static.igem.wiki/teams/4816/wiki/lab-picture.png)

**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](https://static.igem.wiki/teams/4816/wiki/table-aerobic-denitrifiers.jpg)

**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](https://static.igem.wiki/teams/4816/wiki/draft-workflow.png)

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

![pET-23a (+) plasmid scheme](https://static.igem.wiki/teams/4816/wiki/scheme-pet23aplus.png)

**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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27basic-parts%27%29%20%7D%7D)

* napA forward: [BBa\_K4816001](http://parts.igem.org/Part%3ABBa_K4816001)
* napA reverse: [BBa\_K4816002](http://parts.igem.org/Part%3ABBa_K4816002)
* nirS forward: [BBa\_K4816003](http://parts.igem.org/Part%3ABBa_K4816003)
* nirS reverse: [BBa\_K4816004](http://parts.igem.org/Part%3ABBa_K4816004)
* norB forward: [BBa\_K4816005](http://parts.igem.org/Part%3ABBa_K4816005)
* norB reverse: [BBa\_K4816008](http://parts.igem.org/Part%3ABBa_K4816008)
* nosZ forward: [BBa\_K4816006](http://parts.igem.org/Part%3ABBa_K4816006)
* nosZ reverse: [BBa\_K4816007](http://parts.igem.org/Part%3ABBa_K4816007)

---

## Gene Documentation

[Here](https://static.igem.wiki/teams/4816/wiki/enzyme-documentation/denitrification-enzyme-structures.pdf), 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.

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## What should this page contain?

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Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

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* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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## Goal: Amplifying Nap A, NirS, NorB and NosZ genes to create our construct

To enhance the overexpression of our target genes in E. coli, our initial step involved the isolation of these
genes, a crucial phase in our project's progression. This isolation process was executed meticulously, beginning
with the design of primers tailored to the specific genes of interest. Subsequently, we embarked on the gene
amplification journey, employing the Polymerase Chain Reaction (PCR) method.

It's worth noting that prior to initiating the PCR amplification, we conducted a thorough in-silico analysis,
optimizing primer sequences for the intended genes. However, our actual PCR outcomes proved to be somewhat less
promising than anticipated, prompting a deeper examination of our procedures.

Despite adhering to the recommended annealing temperatures provided by both SnapGene and NCBI Primer Blast, our
PCR results fell short of our expectations. For instance, consider the case of the NapA gene, for which our
in-silico analysis suggested an ideal annealing temperature of 57℃. Yet, in practice, the resulting PCR band
exceeded our desired size, measuring larger than 10 kb, as visually depicted in Figure 1.

![pET-23a (+) plasmid scheme](https://static.igem.wiki/teams/4816/wiki/gel-electrophoresis-1.png)

**Figure 1.** Gel electrophoresis of NapA PCR product, with annealing temperature at 57 degrees Celsius.

This discrepancy between our in-silico predictions and the experimental outcomes raised important questions and
challenges that demanded our attention and further investigation to optimize our gene isolation. After conducting
numerous gradient experiments and exploring alternative polymerases, we successfully determined the optimal
annealing temperatures for each primer. However, we encountered a challenge where the resulting bands are not
consistently robust, leading to low-concentration DNA. This limitation has hindered our ability to proceed with
the subsequent step of inserting the isolated DNA into the vector.

![pET-23a (+) plasmid scheme](https://static.igem.wiki/teams/4816/wiki/gel-electrophoresis-2.png)

**Figure 2.** Gel electrophoresis of PCR products, with corresponding primers’ ideal annealing temperatures.

---

## Future Direction

Despite our thorough in-silico analysis, we've faced challenges in amplifying certain genes as expected. Our next step involves redesigning primers,
particularly for the NapA gene, which encodes nitrate reductase and is pivotal in the denitrification pathway. We aim to enhance primer specificity
and efficiency. Our experiences, though challenging, are valuable lessons for future iGEM teams, who can learn from our journey, including the
complexities of in-silico designs versus real-world lab work. We hope to contribute insights into primer design and gene amplification for the benefit
of future projects. Additionally, we'll work to optimize the entire gene amplification process, ensuring we collect sufficient genetic material for
subsequent steps like restriction digests. Our commitment to improvement and sharing knowledge remains unwavering as we advance in the field of genetic
engineering and synthetic biology.

## Next Steps

To envision the continuation of our project, let's consider the essential steps that lie ahead. First and foremost, we need to address the challenge
of gene amplification. One potential approach might involve a redesign of the primers, particularly for the NapA gene. However, it's crucial to tread
carefully, as altering the primer sequence could inadvertently lead to an undesirable increase in the lengths of the flanked genes, a complication
we'd prefer to avoid.

For the other three genes, we've successfully identified their ideal annealing temperatures. Yet, despite this progress, the yield of DNA remains
insufficient for our requirements. To overcome this hurdle, our next course of action would involve increasing the DNA concentration. In an ideal
scenario, we would implement a precipitation technique with ethanol, which holds the promise of significantly boosting our DNA yield.

Upon achieving successful gene amplification, our project could move forward as [originally planned](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D). This includes the insertion of the genes of
interest into plasmids and the subsequent transformation of our E. coli BL21 strain. The ultimate goal here is to achieve a higher yield of the
enzymes that are central to our research. Following the successful expression and harvesting of cells, the purification process would come into play.

In parallel to our experimental work, modeling the enzymatic activities would be a crucial aspect of our project. We would aim to model the activity
of the four enzymes, each equipped with their histags. Furthermore, we would explore the activity of these enzymes when connected to a peptide linker
with the supply of electrons. It would be imperative during this process to ensure that the active sites of the enzymes remain unobstructed for
optimal functionality.

As we progress and set up our bioreactor, a field test with available groundwater would mark a significant milestone. This field test would provide
invaluable insights into the efficiency of our bioreactor in denitrifying water under real-world conditions. Only after the successful completion
of this field test could we begin contemplating the design of a scaled-up bioreactor, a prospect that holds immense potential for practical applications.

In summary, while these steps represent the envisioned progression of our project, it's important to acknowledge that we are at the stage of planning
and preparation, with much work ahead of us to transform these ideas into tangible results.

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