iGEM 2021 — Team Wageningen_UR

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# Team:Wageningen UR/Notebook

iGEM Wageningen 2021

[![logo](https://static.igem.org/mediawiki/2021/f/f0/T--Wageningen_UR--Cattlelyst_logo.png "Logo")](https://2021.igem.org/Team%3AWageningen_UR)
*menu*

* [![logo](https://static.igem.org/mediawiki/2021/2/27/T--Wageningen_UR--Cattlelyst_logo_small.png "Logo")](https://2021.igem.org/Team%3AWageningen_UR)
* Project*arrow\_drop\_down*

+ [Description](https://2021.igem.org/Team%3AWageningen_UR/Description)
+ [Engineering](https://2021.igem.org/Team%3AWageningen_UR/Engineering)
+ [Background](https://2021.igem.org/Team%3AWageningen_UR/Background)
+ [Implementation](https://2021.igem.org/Team%3AWageningen_UR/Implementation)
+ [Sustainable development goals](https://2021.igem.org/Team%3AWageningen_UR/Sustainable)
+ [Contribution](https://2021.igem.org/Team%3AWageningen_UR/Contribution)

* Results*arrow\_drop\_down*

+ [Overview](https://2021.igem.org/Team%3AWageningen_UR/Results)
+ [Modeling](https://2021.igem.org/Team%3AWageningen_UR/Model)
+ [Wetlab](https://2021.igem.org/Team%3AWageningen_UR/Wetlab)
+ [Achievements](https://2021.igem.org/Team%3AWageningen_UR/Achievements)
+ [Notebook](https://2021.igem.org/Team%3AWageningen_UR/Notebook)

* Bioparts*arrow\_drop\_down*

+ [The iGEM PIPE](https://2021.igem.org/Team%3AWageningen_UR/Software)
+ [Wikibase](https://2021.igem.org/Team%3AWageningen_UR/Wikibase)
+ [Biobricks](https://2021.igem.org/Team%3AWageningen_UR/Parts)

* Human Practices*arrow\_drop\_down*

+ [Integrated Human Practices](https://2021.igem.org/Team%3AWageningen_UR/Human_Practices)
+ [Communication](https://2021.igem.org/Team%3AWageningen_UR/Communication)
+ [Education](https://2021.igem.org/Team%3AWageningen_UR/Education)
+ [Collaborations](https://2021.igem.org/Team%3AWageningen_UR/Collaborations)
+ [Dutch Meetup](https://2021.igem.org/Team%3AWageningen_UR/DutchMeetup)
+ [Lab Olympics](https://2021.igem.org/Team%3AWageningen_UR/LabOlympics)

* Safety*arrow\_drop\_down*

+ [Safety by design](https://2021.igem.org/Team%3AWageningen_UR/SafetyByDesign)
+ [Lab safety](https://2021.igem.org/Team%3AWageningen_UR/Safety)
+ [Ethics](https://2021.igem.org/Team%3AWageningen_UR/Ethics)

* About us*arrow\_drop\_down*

+ [Team](https://2021.igem.org/Team%3AWageningen_UR/Team)
+ [Our journey](https://2021.igem.org/Team%3AWageningen_UR/OurJourney)
+ [Attributions](https://2021.igem.org/Team%3AWageningen_UR/Attributions)
+ [Sponsors](https://2021.igem.org/Team%3AWageningen_UR/Sponsors)

* [![logo](https://static.igem.org/mediawiki/2021/f/f0/T--Wageningen_UR--Cattlelyst_logo.png "Logo")](https://2021.igem.org/Team%3AWageningen_UR)
  [*clear*](#!)
* + Project*arrow\_drop\_down*

    - [Description](https://2021.igem.org/Team%3AWageningen_UR/Description)
    - [Engineering](https://2021.igem.org/Team%3AWageningen_UR/Engineering)
    - [Background](https://2021.igem.org/Team%3AWageningen_UR/Background)
    - [Sustainable development goals](https://2021.igem.org/Team%3AWageningen_UR/Sustainable)
    - [Contribution](https://2021.igem.org/Team%3AWageningen_UR/Contribution)
    - [Implementation](https://2021.igem.org/Team%3AWageningen_UR/Implementation)
  + Results*arrow\_drop\_down*

    - [Overview](https://2021.igem.org/Team%3AWageningen_UR/Results)
    - [Modeling](https://2021.igem.org/Team%3AWageningen_UR/Model)
    - [Wetlab](https://2021.igem.org/Team%3AWageningen_UR/Wetlab)
    - [Achievements](https://2021.igem.org/Team%3AWageningen_UR/Achievements)
    - [Notebook](https://2021.igem.org/Team%3AWageningen_UR/Notebook)
  + Bioparts*arrow\_drop\_down*

    - [The iGEM PIPE](https://2021.igem.org/Team%3AWageningen_UR/Software)
    - [Wikibase](https://2021.igem.org/Team%3AWageningen_UR/Wikibase)
    - [Biobricks](https://2021.igem.org/Team%3AWageningen_UR/Parts)
  + Human Practices*arrow\_drop\_down*

    - [Integrated Human Practices](https://2021.igem.org/Team%3AWageningen_UR/Human_Practices)
    - [Communication](https://2021.igem.org/Team%3AWageningen_UR/Communication)
    - [Education](https://2021.igem.org/Team%3AWageningen_UR/Education)
    - [Collaborations](https://2021.igem.org/Team%3AWageningen_UR/Collaborations)
    - [Dutch Meetup](https://2021.igem.org/Team%3AWageningen_UR/DutchMeetup)
    - [Lab Olympics](https://2021.igem.org/Team%3AWageningen_UR/LabOlympics)
  + Safety*arrow\_drop\_down*

    - [Safety by design](https://2021.igem.org/Team%3AWageningen_UR/SafetyByDesign)
    - [Lab safety](https://2021.igem.org/Team%3AWageningen_UR/Safety)
    - [Ethics](https://2021.igem.org/Team%3AWageningen_UR/Ethics)
  + About us*arrow\_drop\_down*

    - [Team](https://2021.igem.org/Team%3AWageningen_UR/Team)
    - [Our journey](https://2021.igem.org/Team%3AWageningen_UR/OurJourney)
    - [Attributions](https://2021.igem.org/Team%3AWageningen_UR/Attributions)
    - [Sponsors](https://2021.igem.org/Team%3AWageningen_UR/Sponsors)

# Notebook

![Engineering Photo](https://static.igem.org/mediawiki/2021/1/15/T--Wageningen_UR--Parallax_notebook.jpg)

Curious to know more about our lab life and how we built our models and software tools? On this page you can find a short description of our projects and links to our notebooks. Enjoy reading!

## Wetlab

![subproject hexbadge](https://static.igem.org/mediawiki/2021/0/04/T--Wageningen_UR--Icon_nitrification.png)

##### Synthetic nitrification in *Pseudomonas putida*

Nitrification genes originating from autotrophic nitrifier Nitrosomonas europaea were expressed in *Pseudomonas putida*.
This would allow *P. putida* to convert ammonia to hydroxylamine and subsequently nitrite, the first steps in the conversion of ammonia
to dinitrogen in our biofilter.

[Notebook Thomas](https://2021.igem.org/Team%3AWageningen_UR/Notebook/Thomas)

![subproject hexbadge](https://static.igem.org/mediawiki/2021/e/e1/T--Wageningen_UR--Icon_denitrification.png)

##### Synthetic denitrification in *Pseudomonas putida* (1)

Denitrification genes originating from several native denitrifiers were expressed in *Pseudomonas putida*.
This would allow *P. putida* to convert nitrate or nitrite to dinitrogen gas via the intermediates nitric oxide and nitrous oxide.
These are the final steps in the conversion of ammonia to dinitrogen in our biofilter.

[Notebook Anemoon](https://2021.igem.org/Team%3AWageningen_UR/Notebook/Anemoon)

![subproject hexbadge](https://static.igem.org/mediawiki/2021/e/e1/T--Wageningen_UR--Icon_denitrification.png)

##### Synthetic denitrification in *Pseudomonas putida* (2)

Denitrification genes originating from several native denitrifiers were expressed in *Pseudomonas putida*.
This would allow *P. putida* to convert nitrate or nitrite to dinitrogen gas via the intermediates nitric oxide and nitrous oxide.
These are the final steps in the conversion of ammonia to dinitrogen in our biofilter.

[Notebook Sophieke](https://2021.igem.org/Team%3AWageningen_UR/Notebook/Sophieke)

![subproject hexbadge](https://static.igem.org/mediawiki/2021/a/ae/T--Wageningen_UR--Icon_nitrous.png)

##### Limiting nitrous oxide production in *Pseudomonas putida*

Steady nitrogen conversions were ensured by redirecting the electron flux towards the denitrification machinery in *Pseudomonas putida*. This was
realized by creating denitrification machinery and downregulating native *P. putida*'s ability to respire with oxygen, in parallel.

[Notebook Sanne](https://2021.igem.org/Team%3AWageningen_UR/Notebook/Sanne)

![subproject hexbadge](https://static.igem.org/mediawiki/2021/e/e2/T--Wageningen_UR--Icon_methaneConvers.png)

##### Synthetic methane oxidation in *Escherichia coli* (1)

The heterologous expression of soluble and particulate methane monooxygenase, and the expression of methanoldehydrogenase, completing
the C1 conversion pathway from methane to biomass and carbondioxide in C1 auxotrophic *Escherichia coli strains*.

[Notebook Riemer](https://2021.igem.org/Team%3AWageningen_UR/Notebook/Riemer)

![subproject hexbadge](https://static.igem.org/mediawiki/2021/e/e2/T--Wageningen_UR--Icon_methaneConvers.png)

##### Synthetic methane oxidation in *Escherichia coli* (2)

The heterologous expression of soluble and particulate methane monooxygenase, and the expression of methanoldehydrogenase, completing
the C1 conversion pathway from methane to biomass and carbondioxide in C1 auxotrophic *Escherichia coli strains*.

[Notebook Morris](https://2021.igem.org/Team%3AWageningen_UR/Notebook/Morris)

![subproject hexbadge](https://static.igem.org/mediawiki/2021/5/5e/T--Wageningen_UR--Icon_methaneKill.png)

##### Methane-dependent kill switch in *Escherichia coli*

This kill switch couples methane concentration to a toxin-antitoxin system in *Escherichia coli*. Should the bacterium escape the biofilter, where the methane
concentration is lower, toxin production is activated, thereby killing the cells.

[Notebook Sophie](https://2021.igem.org/Team%3AWageningen_UR/Notebook/Sophie)

![subproject hexbadge](https://static.igem.org/mediawiki/2021/6/68/T--Wageningen_UR--Icon_proximity.png)

##### Proximity-dependent kill switch in a microbial coculture

This kill switch will kill *Pseudomonas putida* and *Escherichia coli* if they escape from the biofilter. Outside the biofilter they lose the close proximity of
each other, activating the kill switch.

[Notebook Jenny](https://2021.igem.org/Team%3AWageningen_UR/Notebook/Jenny)

![subproject hexbadge](https://static.igem.org/mediawiki/2021/9/9d/T--Wageningen_UR--Icon_auxotrophy.png)

##### Bacterial co-dependency

A co-dependency was established between *Escherichia coli* and *Pseudomonas putida* by creating a cross-feeding community. The cross-feeding
is based on amino acid exchange/auxotrophy and a carbon-source dependency.

[Notebook Marta](https://2021.igem.org/Team%3AWageningen_UR/Notebook/Marta)

[Click here to read the protocols](https://2021.igem.org/Team%3AWageningen_UR/Notebook/Protocols)

## Modeling

![subproject hexbadge](https://static.igem.org/mediawiki/2021/8/8c/T--Wageningen_UR--icon_modeling_nitrogen.png)

##### Modeling Coupled Nitrification and Aerobic Denitrification

To increase our conceptual understanding of the HNAD phenomenon, we developed a dynamic model. This model is able to simulate nitrogen dynamics connected to the increase in cell population.

[Notebook Sanne](https://2021.igem.org/Team%3AWageningen_UR/Notebook/Sanne)

![subproject hexbadge](https://static.igem.org/mediawiki/2021/4/44/T--Wageningen_UR--icon_modeling_safety.png)

##### Modeling a formaldehyde-dependent toxin-antitoxin system

To gain understanding of the dynamics of the methane-based biosafety circuit, a model was built using ordinary differential equations. Hypothetical systems incorporating protein X and a competitor protein were created to improve formaldehyde sensitivity.

[Notebook Jenny](https://2021.igem.org/Team%3AWageningen_UR/Notebook/Jenny)

![subproject hexbadge](https://static.igem.org/mediawiki/2021/e/e5/T--Wageningen_UR--icon_modeling_biofilter.png)

##### Modeling biofilter performance

To investigate the combined reduction of methane and ammonia emissions, a biofilter model was developed that determines the reactor size and performance for the microbial culture options we have considered for Cattlelyst. The model combines metabolic and reactor modeling with three different scales based on mass balances: single cell, single particle, and reactor scale.

[Notebook Anemoon](https://2021.igem.org/Team%3AWageningen_UR/Notebook/Anemoon)

## Software

![subproject hexbadge](https://static.igem.org/mediawiki/2021/c/c8/T--Wageningen_UR--PIPE-logo.png)

##### The iGEM PIPE

Lorem ipsum dolor sit amet consectetur, adipisicing elit. Distinctio soluta ipsa voluptates ad quae repellendus voluptate quis. Officia, commodi pariatur!.

[Notebook Delielena](https://2021.igem.org/Team%3AWageningen_UR/Notebook/Delielena)

![subproject hexbadge](https://static.igem.org/mediawiki/2021/c/c8/T--Wageningen_UR--PIPE-logo.png)

##### Wikibase

Creation of Semantic Database to comprehensively search The iGEM Registry of Standard Biological Parts.

[Notebook Riemer](https://2021.igem.org/Team%3AWageningen_UR/Notebook/Riemer)

##### About Cattlelyst

Cattlelyst is the name of the iGEM 2021 WUR team. Our name is a mix of 1) our loyal furry friends, cattle, and 2) catalyst, which is something that increases the rate of a reaction.
We are developing “the something” that converts the detrimental gaseous emissions of cattle, hence our name Cattlelyst.

Are you curious about our journey? We have written about our adventures in our blog, which you can find here:

##### Keep in touch!

* igem@wur.nl
* [iGEMWageningen](https://www.facebook.com/iGEMWageningen/?hc_ref=ARTIdtcStpEkeL1jRvNQs5XrYIZk9-Nl-Ieh6o3DXz7A9PsWjnip9gC1W0BEGBf_9Rg&fref=nf&__tn__=kC-R)
* [@igemwageningen](https://twitter.com/igemwageningen?ref_src=twsrc%5Egoogle%7Ctwcamp%5Eserp%7Ctwgr%5Eauthor)
* [iGEM Wageningen](https://nl.linkedin.com/company/igem-wageningen)
* [igem.wageningen](https://www.instagram.com/igem.wageningen/)

![logo](https://static.igem.org/mediawiki/2021/f/f0/T--Wageningen_UR--Cattlelyst_logo.png "Logo")

*format\_list\_bulleted*

* ###### SECTIONS
* [*arrow\_upward*](#home)

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# Team:Wageningen UR/Engineering

iGEM Wageningen 2021

[![logo](https://static.igem.org/mediawiki/2021/f/f0/T--Wageningen_UR--Cattlelyst_logo.png "Logo")](https://2021.igem.org/Team%3AWageningen_UR)
*menu*

* [![logo](https://static.igem.org/mediawiki/2021/2/27/T--Wageningen_UR--Cattlelyst_logo_small.png "Logo")](https://2021.igem.org/Team%3AWageningen_UR)
* Project*arrow\_drop\_down*

+ [Description](https://2021.igem.org/Team%3AWageningen_UR/Description)
+ [Engineering](https://2021.igem.org/Team%3AWageningen_UR/Engineering)
+ [Background](https://2021.igem.org/Team%3AWageningen_UR/Background)
+ [Implementation](https://2021.igem.org/Team%3AWageningen_UR/Implementation)
+ [Sustainable development goals](https://2021.igem.org/Team%3AWageningen_UR/Sustainable)
+ [Contribution](https://2021.igem.org/Team%3AWageningen_UR/Contribution)

* Results*arrow\_drop\_down*

+ [Overview](https://2021.igem.org/Team%3AWageningen_UR/Results)
+ [Modeling](https://2021.igem.org/Team%3AWageningen_UR/Model)
+ [Wetlab](https://2021.igem.org/Team%3AWageningen_UR/Wetlab)
+ [Achievements](https://2021.igem.org/Team%3AWageningen_UR/Achievements)
+ [Notebook](https://2021.igem.org/Team%3AWageningen_UR/Notebook)

* Bioparts*arrow\_drop\_down*

+ [The iGEM PIPE](https://2021.igem.org/Team%3AWageningen_UR/Software)
+ [Wikibase](https://2021.igem.org/Team%3AWageningen_UR/Wikibase)
+ [Biobricks](https://2021.igem.org/Team%3AWageningen_UR/Parts)

* Human Practices*arrow\_drop\_down*

+ [Integrated Human Practices](https://2021.igem.org/Team%3AWageningen_UR/Human_Practices)
+ [Communication](https://2021.igem.org/Team%3AWageningen_UR/Communication)
+ [Education](https://2021.igem.org/Team%3AWageningen_UR/Education)
+ [Collaborations](https://2021.igem.org/Team%3AWageningen_UR/Collaborations)
+ [Dutch Meetup](https://2021.igem.org/Team%3AWageningen_UR/DutchMeetup)
+ [Lab Olympics](https://2021.igem.org/Team%3AWageningen_UR/LabOlympics)

* Safety*arrow\_drop\_down*

+ [Safety by design](https://2021.igem.org/Team%3AWageningen_UR/SafetyByDesign)
+ [Lab safety](https://2021.igem.org/Team%3AWageningen_UR/Safety)
+ [Ethics](https://2021.igem.org/Team%3AWageningen_UR/Ethics)

* About us*arrow\_drop\_down*

+ [Team](https://2021.igem.org/Team%3AWageningen_UR/Team)
+ [Our journey](https://2021.igem.org/Team%3AWageningen_UR/OurJourney)
+ [Attributions](https://2021.igem.org/Team%3AWageningen_UR/Attributions)
+ [Sponsors](https://2021.igem.org/Team%3AWageningen_UR/Sponsors)

* [![logo](https://static.igem.org/mediawiki/2021/f/f0/T--Wageningen_UR--Cattlelyst_logo.png "Logo")](https://2021.igem.org/Team%3AWageningen_UR)
  [*clear*](#!)
* + Project*arrow\_drop\_down*

    - [Description](https://2021.igem.org/Team%3AWageningen_UR/Description)
    - [Engineering](https://2021.igem.org/Team%3AWageningen_UR/Engineering)
    - [Background](https://2021.igem.org/Team%3AWageningen_UR/Background)
    - [Sustainable development goals](https://2021.igem.org/Team%3AWageningen_UR/Sustainable)
    - [Contribution](https://2021.igem.org/Team%3AWageningen_UR/Contribution)
    - [Implementation](https://2021.igem.org/Team%3AWageningen_UR/Implementation)
  + Results*arrow\_drop\_down*

    - [Overview](https://2021.igem.org/Team%3AWageningen_UR/Results)
    - [Modeling](https://2021.igem.org/Team%3AWageningen_UR/Model)
    - [Wetlab](https://2021.igem.org/Team%3AWageningen_UR/Wetlab)
    - [Achievements](https://2021.igem.org/Team%3AWageningen_UR/Achievements)
    - [Notebook](https://2021.igem.org/Team%3AWageningen_UR/Notebook)
  + Bioparts*arrow\_drop\_down*

    - [The iGEM PIPE](https://2021.igem.org/Team%3AWageningen_UR/Software)
    - [Wikibase](https://2021.igem.org/Team%3AWageningen_UR/Wikibase)
    - [Biobricks](https://2021.igem.org/Team%3AWageningen_UR/Parts)
  + Human Practices*arrow\_drop\_down*

    - [Integrated Human Practices](https://2021.igem.org/Team%3AWageningen_UR/Human_Practices)
    - [Communication](https://2021.igem.org/Team%3AWageningen_UR/Communication)
    - [Education](https://2021.igem.org/Team%3AWageningen_UR/Education)
    - [Collaborations](https://2021.igem.org/Team%3AWageningen_UR/Collaborations)
    - [Dutch Meetup](https://2021.igem.org/Team%3AWageningen_UR/DutchMeetup)
    - [Lab Olympics](https://2021.igem.org/Team%3AWageningen_UR/LabOlympics)
  + Safety*arrow\_drop\_down*

    - [Safety by design](https://2021.igem.org/Team%3AWageningen_UR/SafetyByDesign)
    - [Lab safety](https://2021.igem.org/Team%3AWageningen_UR/Safety)
    - [Ethics](https://2021.igem.org/Team%3AWageningen_UR/Ethics)
  + About us*arrow\_drop\_down*

    - [Team](https://2021.igem.org/Team%3AWageningen_UR/Team)
    - [Our journey](https://2021.igem.org/Team%3AWageningen_UR/OurJourney)
    - [Attributions](https://2021.igem.org/Team%3AWageningen_UR/Attributions)
    - [Sponsors](https://2021.igem.org/Team%3AWageningen_UR/Sponsors)

# Engineering

![Engineering Photo](https://static.igem.org/mediawiki/2021/3/37/T--Wageningen_UR--Parallax_engineering.jpg)

Driven to provide a solution to the harmful ammonia and methane emissions from cattle, we invented Cattlelyst. After extensive research and reaching out to various  [stakeholders](https://2021.igem.org/Team%3AWageningen_UR/Human_Practices), we decided that Cattlelyst would be a biofilter
containing bacteria that can convert these gasses into harmless compounds. We further built on this idea by designing our biological system, which is divided into three pillars: 1. Methane conversion,
2. Ammonia conversion and 3. Safety. For each of these pillars we went through one or multiple engineering cycles (**Research** - **Design** – **Build** – **Test** – **Learn**) until our final design.

 We began by
researching the required conversions of methane (CH4) into carbon dioxide (CO2) and ammonia (NH3) into dinitrogen gas (N2) and how they are performed in
nature. More information about this can be found in [Background](https://2021.igem.org/Team%3AWageningen_UR/Background#Microorganisms). Next, we explored candidate bacteria for our biofilter. In this page we will discuss why we chose *Escherichia coli* (*E. coli*)
and *Pseudomonas putida* (*P. Putida*). Next, we will discuss how we went through the engineering cycles to design, build, test and learn from methane and ammonia conversions in these organisms and the
accompanying safety system of our project.

## Chassis bacteria for co-cultures

While designing the biofilter we came across several requirements for candidate bacteria. The biofilter will be situated in an aerobic environment, so the bacteria should be well adapted for living in
such an environment. Additionally, as we aim to reduce methane as well as ammonia emissions, both microbial conversions should happen. There is not any known bacterium capable of performing both
conversions, and natural organisms with the required pathways are not equipped to perform the conversions in Cattlelyst, which will be explained further down. Therefore, bacterial chassis are needed in which the pathways
can be engineered. These bacteria should be non-pathogenic and genetically accessible. After extensively reading relevant literature and orientating ourselves on possible mechanisms we have come up with
two candidate bacteria that will be the chassis for our project.

* [*Escherichia coli*](#tab1)
* [*Pseudomonas putida*](#tab2)

The natural methanotroph *Methylococcus capsulatus* is one of the, if not the, best studied methanotroph. It harbors the complete pathway for converting methane into biomass and carbon
dioxide and contains both the common soluble methane monooxygenase (sMMO) as well as the uncommon particulate methane monooxygenase (pMMO). More information about these enzymes can be found
in [Background](https://2021.igem.org/Team%3AWageningen_UR/Background#Microorganisms). Although the bacterium is also able to nitrify, no genes are predicted to perform denitrification in aerobic
conditions. Unfortunately, *Methylococcus capsulatus* is laborious to manipulate, as the genetic tools are very limited and the growth rate is very low, resulting in culturing times of 3 to 7 days
[1], [2]. Since we could not engineer a natural methanotroph, we could not engineer ammonia conversion into dinitrogen gas. Of course, *M. capsulatus* can be used as a source of inspiration for its
genes and methane metabolism. For instance, the genes encoding for pMMO, sMMO and methanol dehydrogenase (Mdh) are of interest to our project.

Therefore, the genetically accessible model organism *E. coli* was chosen as chassis to establish the methane conversion pathway. This bacterium naturally does not contain the methane or
ammonia conversion pathway. However, the Academia Sinica lab of the Institute of Biological Chemistry in Taipai, Taiwan has engineered an *E. coli* strain that can grow fully on methanol,
the first compound in the oxidation pathway of methane. Thus, only the first conversion from methane to methanol needs to be engineered in this strain [3]. Initially, we wanted to establish both
methane and ammonia conversion in *E. coli* alone. We called this the mono-culture approach.

![](https://static.igem.org/mediawiki/2021/5/5c/T--Wageningen_UR--ecoli_backg.png)

Although specific microorganisms that perform Heterotrophic Nitrification Aerobic Denitrification (HNAD) exist, the conditions for which these organisms evolved to are
highly specific. A plethora of external factors: carbon source, temperature, dissolved oxygen, carbon/nitrogen ratio influence pathway efficiency greatly [4], [5], [6]. Moreover, the
extent of the impact of these factors is not ubiquitous among the different HNAD bacteria. Given that the application for Cattlelyst differs from nature’s purposes, the rationale to develop the HNAD system,
by means of synthetic biology, to fit our application was born.

The non-denitrifying organism *P. putida* was chosen as chassis as it phylogenetically related to the HNAD harboring bacteria *P. stutzeri* and *P. aeruginosa*. Given the short
evolutionary distance, it was reasoned that minimal adjustments were to be made to heterologously express the HNAD pathway. *P. putida* is recognized as a model laboratory bacterium
due to its ability to adapt to harsh environmental conditions, fast growth, simple nutritional requirements and an extensive genetic toolbox [7].

![](https://static.igem.org/mediawiki/2021/4/4b/T--Wageningen_UR--putida-backg.png)

#### Co-culture

In some instances, genes can be toxic to bacteria. This can lead to gene alterations and unsuccessful expression of proteins. This was seen for some membrane proteins in *E. coli*, heterologous
expression of these proteins killed  *E. coli* [8]. In the ammonia conversion pathway specifically, ammonia monooxygenase (AMO) and nitric oxide reductase (Nor) are membrane proteins. Thus, to
spread our chances, we wanted to in parallel develop ammonia conversion in *P. putida*. Given that parts of the methane oxidation pathway have been successfully established in *E. coli*,
we did not develop this in *P. putida* [3], [9].

Having the metabolic pathways of methane and ammonia conversion divided over the two bacterial candidates paved the way for a having a co-culture, with *E. coli* serving as the methanotroph and *P. putida* as the ammoniatroph. Moreover, by having a co-culture the growth of both bacteria could be improved. By creating a cross-feeding interaction between the bacteria, metabolic labor could be divided, leading to advantages in growth [10]. Next to engineering *E. coli* and *P. putida* to contain the methane and ammonia conversion pathway, respectively, we therefore also needed to establish cross-feeding interactions to create a stable and effective co-culture.

In the following chapter, we will discuss how we went through the engineering cycles to design, build, test and learn methane and ammonia conversion in *P. putida* and *E.coli*, and the
accompanying safety system of our project. This safety system included creating an amino acid auxotrophy and carbon source dependency between the bacteria to achieve a successful and safe co-culture.

![](https://static.igem.org/mediawiki/2021/4/47/T--Wageningen_UR--IHP_GMOdealbreaker.png)

In some instances, genes can be toxic to bacteria. This can lead to gene alterations and unsuccessful expression of proteins. This was seen for some membrane proteins in *E. coli*, heterologous expression of these proteins killed  *E. coli* [8]. In the ammonia conversion pathway specifically, ammonia monooxygenase (AMO) and nitric oxide reductase are membrane proteins. Thus, to spread our chances, we wanted to in parallel develop ammonia conversion in *P. putida*. Given that parts of the methane oxidation pathway have been successfully established in *E. coli*, we did not develop this in *P. putida* [3], [9].

Having the metabolic pathways of methane and ammonia conversion divided over the two bacterial candidates paved the way for a having a co-culture, with *E. coli* serving as the methanotroph
and *P. putida* as the ammoniatroph. Moreover, by having a co-culture the growth of both bacteria could be improved. By creating a cross-feeding interaction between the bacteria, metabolic
labor could be divided, leading to advantages in growth [10]. Next to engineering *E. coli* and *P. putida* to contain the methane and ammonia conversion pathway, respectively, we therefore
also needed to establish cross-feeding interactions to create a stable and effective co-culture.

In the following chapter, we will discuss how we went through the engineering cycles to design, build and test methane and ammonia conversion in *P. putida* and *E.coli*, and the
accompanying safety system of our project. This safety system included creating an amino acid auxotrophy and carbon source dependency between the bacteria to achieve a successful and safe co-culture.

![](https://static.igem.org/mediawiki/2021/4/47/T--Wageningen_UR--IHP_GMOdealbreaker.png)

## Engineering the co-culture

Having selected the bacterial chassis for the methane and ammonia conversions in Cattlelyst, we needed to design our approach for building in these pathways and making them work. As mentioned above, we
have organized our project into three pillars: 1. Methane conversion, 2 Ammonia conversion and 3. Safety. During the next chapters we will discuss the engineering cycles we have gone through with
each pillar during our project

![Icon ammonia](https://static.igem.org/mediawiki/2021/5/50/T--Wageningen_UR--Icon_ammonia.png)

### Ammonia removal

![Icon ammonia](https://static.igem.org/mediawiki/2021/5/50/T--Wageningen_UR--Icon_ammonia.png)

### Ammonia removal

#### *Cycle 1*

**Design**
For our biofilter, we wanted to convert ammonia into dinitrogen gas. In the initial research phase, we read about newly-discovered bacteria that can complete this conversion aerobically, which is
perfect for its application in Cattlelyst. The mechanism they employ is referred to as coupled heterotrophic nitrification-aerobic denitrification, in short HNAD. Unfortunately, HNAD bacteria
have evolved to survive in completely different conditions to ours. Therefore, we needed to engineer the HNAD pathway into a microbe that does fit our conditions. At this moment, the exact mechanism
explaining the conversion is poorly understood. To gain insight in the HNAD phenomenon and build the groundwork for our future wet lab projects, we decided to develop a dynamic model. More explanation
can be found on the [nitrogen conversion model page](https://2021.igem.org/Team%3AWageningen_UR/Model/Nitrogen).

**Build**
First, the model structure was established based on pathways suggested by Wehrfritz et al. [11] and Joo et al. [12]. published experimental data for *Pseudomonas stutzeri* strains YZN-001
[13] and XL-2 [4], and (non-)enzymatic conversions reported by Caranto et al. [14]. Then, the mechanism was represented mathematically as a system of ordinary differential equations (ODEs).
Subsequently, the model was collectively fit to agree with time-series data for *Pseudomonas stutzeri* strains YZN-001 [13] and XL-2, validating this approach [4].

**Test**
The final model could capture the nitrogen dynamics for *P. stutzeri* strains YZN-001 [13] and XL-2 [4] well. We further validated the model by comparing the simulated nitrogen dynamics with
data for **P. putida** ZN1 [15]. Moreover, we found that when the cells are exposed to nitrate, the harmful intermediate nitrous oxide accumulates in the medium. Therefore, we identified
parameters responsible for this accumulation. Lastly, given that the data is obtained in high ammonia conditions, we simulated the system for low ammonia concentrations, to reflect the biofilter
conditions better.

**Learn**
We learned that the model was able to predict nitrogen dynamics for different conditions and different strains. This suggested that the proposed pathway structure underlies the HNAD phenomenon.
Moreover, we learned that with HNAD we can risk to accumulate nitrous oxide. With the model, we identified the maximum conversion rate of nitrous oxide reductase to be most interesting to optimize
for in vivo. Lastly, we learned that the ratio of nitrogen that goes to biomass and HNAD does not change in low ammonia concentrations. This suggests that HNAD could be a growth-coupled process,
which could explain the difference between autotrophic nitrifiers and heterotrophic nitrifiers. However, the HNAD behavior is rarely studied for in low ammonia concentrations, thus more research
is required to validate these model predictions.
![Icon ammonia](https://static.igem.org/mediawiki/2021/b/ba/T--Wageningen_UR--Model_Sanne_simulation_YZN001.png)
***Figure 1:** Model simulation of extracellular N-species for best parameter set. The continuous line corresponds to model simulations, the squares are time-series data. Upper panel details
dynamics for extracellular nitrogen species in condition for *P. stutzeri strain YZN-001*. Pink: [ammonia]ex , Red: [dinitrogen gas]ex, Green: [nitrate]ex,
Blue: [nitrite]ex, Yellow: [nitric oxide]ex, Black (top figure) [Nitrous oxide]ex. Lower panel details dynamics of the population volume in condition for *P. stutzeri*
strain YZN-001; Continuous line: population volume, Squares: data for population volume.*

#### *Cycle 2*

**Design**
After gaining in-depth knowledge on HNAD through modeling, we designed this pathway as such that it could be built into the model organism **P. putida**. A schematic overview of the
pathway we built can be seen in Figure 2 below and more information about it can be found in [Background](https://2021.igem.org/Team%3AWageningen_UR/Background#Microorganisms).

![Icon ammonia](https://static.igem.org/mediawiki/2021/7/7a/T--Wageningen_UR--nitrogen-path.png)
***Figure 2:** Illustration of the pathway for the conversion of ammonia. The orange boxes indicate the enzymes. AMO = ammonia monooxygenase, HAO = Hydroxylamine oxidoreductase, Nap = periplasmic nitrate
reductase, Nir = nitrite reductase, Nor = nitric oxide reductase, Nos = nitrous oxide reductase.*

Since the genes involved in heterotrophic nitrification are not completely clarified yet, we used the genes from the autotrophic nitrifier *Nitrosomonas europea* to design a [synthetic nitrification](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/Nitrification) pathway.
These genes, AMO and HAO, were codon harmonized for heterologous expression in **P. putida**.

For denitrification, multiple approaches were chosen to increase the chances of success. The first encompassed engineering all four denitrification enzymes separately, each with a synthetic regulation.
With this [mosaic approach](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/SyntheticDenitrification%20%20%20%20%20%20%20%20%20%20%20%20%20), the denitrification pathway can be optimized in such a way that no intermediates accumulate. Additionally, to ensure optimal conversion of nitrate/nitrite to dinitrogen gas,
we wanted to compare the activity of enzymes of different native aerobic denitrifiers.

The second approach, called the [plug and play approach](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/ElectronBalance), included introducing the entire denitrification into *P. putida* at once. To prevent nitrous oxide formation, we designed a way to
redirect the electron flux from the aerobic respiration machinery to the denitrification machinery. By rewiring the electron transport system, the likelihood that insufficient electrons are supplied
to the denitrification machinery, which consequently results in nitrous oxide production, will be decreased. To achieve this, a CRISPR interference (CRISPRi) system was designed to downregulate the
terminal oxidases of *P. putida* to reduce expression of terminal oxidases, and whith that reduce the amount of electrons utilized in oxygen respiration.

**Build**
To engineer the enzymes for nitrification and denitrifications separately into *P. putida*, the encoding genes for each enzyme were cloned into a plasmid. To accomplish this, genes for
denitrification from multiple native denitrifiers were used.

The plug and play approach for denitrification was carried out by cloning all the required genes originating from one organism and placing them in a single plasmid, resulting in the construction of
a bacterial artificial chromosome (BAC). Subsequently, the entire denitrification pathway was integrated in the genome of **P. putida**. In parallel, various CRISPRi spacers targeting
the five terminal oxidases were made.

**Test**
Once all the plasmids for nitrification and denitrification were successfully constructed and transformed into **P. putida**. With all four denitrification enzymes, experiments were performed to quantify the activities *in vivo*, by measuring the amount of substrate consumed or the amount of product
made by **P. putida**. The first two enzymes of the denitrification pathway, Nap and Nir, showed the desired activities. Moreover, when combining these enzymes, no harmful intermediates
accumulated anymore, suggesting that the concatenated conversions work (figure 3). When testing the last two enzymes of the pathway, no activity was observed. However, due to time constrains only one
experiment was performed with these enzymes. Unfortunately, due to time constrains the nitrification enzymes
could not be tested.

![](https://static.igem.org/mediawiki/2021/9/9b/T--Wageningen_UR--Wetlab_synDenit_napNir.png)
***Figure 3:** NO2- production by P. putida EM42 ∆nasT containing the nap operon originating from P. denitrificans on plasmid (Nap) or containing the nir operon origination
from P. stutzeri on plasmid as well (Nap + Nir). The NO2- production was corrected for the change in OD over the time span of 24 hours.*

For the plug and play approach, the entire denitrification pathway was tested by quantifying the amount of nitrate that was converted. However, this assay was not sensitive enough to conclude
whether the pathway was active. Therefore, experiments were performed to measure if intermediates of the pathway accumulated. These tests showed that a bit of nitrite and trace amounts of nitrous
oxide accumulated, suggesting that the first three enzymes of the pathway worked. However, more research is needed to derive conclusions on the efficiency of the pathway as a whole.

In-parallel with the plug and play approach, 15 CRISPRi plasmids that downregulate **P. putida**’s terminal oxidases were developed. The effect of downregulating these essential genes
was quantified by comparing growth curves. If downregulation of a terminal oxidase impairs growth significantly, it would be a good candidate to redirect the electron flux towards the
denitrification machinery. All spacers were tested thoroughly,but only for the spacer targeting the promoter of the Cyo oxidase, we found a growth defect during the exponential phase. Measuring the effect of downregulation of multiple terminal
oxidases at the same time should be the next step. However, due to time constraints, a so-called multiplex CRISPRi system could not be developed.

**Learn**
For the mosaic approach of denitrification we showed that the first two enzymes, Nap and Nir, are active separately and combined in **P. putida**, but the Nor and Nos did not work individually
yet. Nor was not yet tested with its accessory proteins due to time constrains. However, there are suggestions that Nor works for the
plug-and-play **P. putida** strain, given that trace elements of nitrous oxide accumulate. This slight accumulation does not exclude the possibility that Nos is active in the plug-and-play
strain. For the mosaic approach, we learned that even though Nos was expressed with accessory proteins, it is still not enough to result in an active enzyme. Lastly, we learned that downregulation
of the promoter of the Cyo oxidase with one CRISPRi spacer could impair growth during the exponential phase. All in all, more research is needed for successful nitrification and denitrification in **P. putida**.

![Icon ammonia](https://static.igem.org/mediawiki/2021/2/22/T--Wageningen_UR--Icon_methane.png)

### Methane Oxidation

![Icon ammonia](https://static.igem.org/mediawiki/2021/2/22/T--Wageningen_UR--Icon_methane.png)

### Methane Oxidation

#### *Cycle 1*

**Design**
As discussed in the Methanotroph section in [Background](https://2021.igem.org/Team%3AWageningen_UR/Background#Microorganisms), the consumption of methane is facilitated by two catalytic reactions. The enzymes sMMO and pMMO both
convert methane into methanol and Mdh subsequently converts methanol into formaldehyde (Figure 4). This formaldehyde can be assimilated into biomass. In this project, we used two *E. coli*
strains used that are capable of this [3], [16] Together with Mdh and pMMO or sMMO these *E. coli* strains are able to either grow solely on methane as carbon source or are auxotrophic for
methane. For a more in-depth explanation, consult [the Wetlab MMO page](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/MMO).

![Icon ammonia](https://static.igem.org/mediawiki/2021/1/1a/T--Wageningen_UR--methane-path-sanne.png)
***Figure 4:** Methane conversion pathway. Methane is converted to methanol via soluble or particulate methane monooxygenase (sMMO/pMMO). Methanol is subsequently converted to formaldehyde via
methanoldehydrogenase (Mdh). Formaldehyde/formate can be incorporated into biomass or converted into carbon dioxide.*

In addition to using literature as a sole source of inspiration, we also used our in-house developed tool [PIPE](https://2021.igem.org/Team%3AWageningen_UR/Software). The iGEM PIPE was used to model the conversion of methane into a high-value compound,
in this case the production of L-lactate, a commercially relevant compound [17]. Our tool also suggested using methane monooxygenase as well as methanol dehydrogenase in *E. coli* , when
this strain was forced to both grow on methane and produce L-lactate (see [the PIPE test case](https://2021.igem.org/Team%3AWageningen_UR/Software/Testcase_validation)).

**Build**
To build these different approaches we started in the lab with two tasks. First, sMMO and pMMO were constructed on plasmids and transformed into *E. coli*. For the pMMO we followed the method
as described in [18] and for the second enzyme, sMMO the building was unsuccessful and the testing phase was not reached. However, this was not essential for the completion of this pilar as we used
a parallel strategy, as pMMO and sMMO are similar in function.

Additionally, we engineered the strain already auxotrophic for formaldehyde to be auxotrophic for methanol, produced by MMO. This was established by incorporating Mdh into the pathway, which
converts methanol into formaldehyde and finally, biomass.

**Test**
The pMMO was tested in two different ways. First it was tested *in vivo* together with duroquinone as additional reducing power. No production of methanol was observed when *E. coli*
containing the pMMO was cultured in sealed bottles with methane in the headspace. Secondly, the pMMO was isolated and tested in an *in vitro* enzyme assay with duroquinone as additional
reducing power [18]. No production of methanol was observed when measuring with GC. We also tested the formaldehyde
auxotrophic strain containing the Mdh construct and we showed that this strain is now able to grow with methanol as sole C1 source. Thus, this strain is now auxotrophic for methanol, which is
produced by MMO.

**Learn**
What was the reason for not being able to clone the sMMO, and why was the pMMO not active? These very important questions remained unanswered as of yet, but there are several possibilities.
One is that the duroquinone was not activated properly. As the protocol used did not contain enough detail to fully replicate it, assumptions had to be made regarding exact quantities.
The cloning method for sMMO required several plasmids and as such, many cloning steps and Gibson assemblies are required. Unfortunately within the span of this project it was not possible
to clone sMMO. Yet, from literature we learnt that because of the high sensitivity of pMMO it is a better fit for our project as we deal with low methane concentrations. Although it is suggested
in literature that pMMO could be expressed recombinantly [18]. this has not been done  *in vivo* , which is a requirement for our biofilter. As such it is important to continue engineering
both pMMO and sMMO.

#### *Cycle 2*

**Design**
After the months spent in the lab and the iGEM competition, we will continue the cloning of sMMO and chaperones and also make some alterations to the pMMO plasmid. The final approach is described
in [this page](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/MMO), but we are confident that both enzymes are great options, each having their own merits and problems. Additionally, upon completion of the successful expression of this
enzyme it will be incorporated in the genome of an C1 growing strain and as such, create a completely synthetic methanotroph.

**Test**
After having recombinantly expressed pMMO in *E. coli*, in addition to testing *in vivo*, we will also retest this version *in vitro* and use additional reductants besides duroquinone.

![Icon safety](https://static.igem.org/mediawiki/2021/7/7e/T--Wageningen_UR--Icon_safety.png)

### Safety

![Icon safety](https://static.igem.org/mediawiki/2021/7/7e/T--Wageningen_UR--Icon_safety.png)

### Safety

#### *Kill-switches: Cycle 1*

**Design**
As we are using GMOs in our biofilter, we must ensure biocontainment. Therefore, we designed multiple safety circuits, which make sure both bacteria are killed outside the biofilter. Read more
about this on the [Safety by design page](https://2021.igem.org/Team%3AWageningen_UR/SafetyByDesign)! We started developing our first safety system in the synthetic methanotroph *E. coli* by basing its survival on the higher methane concentration
inside the biofilter than its surroundings. As there are no methane receptors in the cell, we had to look a bit further down the methane oxidation pathway and found out that *E. coli* can
detect the formaldehyde concentration in the cell sensitively by the FrmR protein [19]. We coupled this to a toxin-antitoxin system to create a system where a low formaldehyde concentration
(i.e. escape from the biofilter) triggers the production of a toxin that kills the cell. We used the hok/sok system, as this works on mRNA basis, lessening the metabolic burden on the cell [20].
A detailed explanation of this system can be found on the [methane-dependent kill switch page](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/MethaneKillSwitch).

**Build**
We started developing the methane-dependent kill switch by making a ‘toy model’ of the system. Using data from literature, we estimated part of the parameters of the full circuit. We then built the
full [model](https://2021.igem.org/Team%3AWageningen_UR/Model/Safety) using ordinary differential equations (ODEs) to test the dynamics of the system. Specifically, we tested if the system is sensitive enough to the small differences in formaldehyde
concentrations in the cell based on the methane concentrations in the real-life situation of the cow stable (Figure 5).

![Icon ammonia](https://static.igem.org/mediawiki/2021/d/d4/T--Wageningen_UR--overview_modeling_safety.png)
***Figure 5:** The biosafety circuit which is supposed to kill the microorganism if the formaldehyde concentration in the cell is low. When the formaldehyde concentration is high, formaldehyde
will bind to the FrmR protein which will then dissociation from the Pfrm promotor, thereby allowing transcription of LacI and antitoxin mRNA. LacI will repress the production of toxin mRNA. When
the formaldehyde concentration drops, there is more free FrmR that binds to the Pfrm promoters. This will inhibit the transcription of the antitoxin and LacI. When there is less LacI present,
the transcription of toxin mRNA will not be inhibited anymore. The organism will then be killed due to the production of the toxin that irreversibly damages the cell membrane of the organism.*

**Test**
We used the model to simulate the dynamics of the methane-dependent kill switch and found out that the formaldehyde sensitivity of the circuit is too low. This means that the difference in formaldehyde
concentrations does not result in a sufficiently high hok/sok ratio to kill the cells if they escape. Therefore, we designed and modeled possible circuit extensions to improve the formaldehyde
sensitivity. We found out that some of these could achieve the desired increase in formaldehyde sensitivity, but not all of them would be possible to build in the lab.

**Learn**
From the model, we learned that the formaldehyde sensitivity of the methane-dependent kill switch needs to be improved to ensure cell death in low formaldehyde conditions. We found out that we need
a second input signal on toxin production in the methane-dependent kill switch to improve the dynamics of the circuit. This can be based on a second characteristic of our biofilter: cell density of
the bacteria. Inside the biofilter, there is a dense co-culture, so the cell density is higher than outside our biofilter.

Another possibility to increase formaldehyde sensitivity is to provide bigger differences in formaldehyde concentrations in the cell. In literature we found that *E. coli* strains with knockouts
of the formaldehyde detoxification pathway can tolerate lower formaldehyde concentrations. Therefore, creating these knock-outs could help in increasing the formaldehyde sensitivity of the
methane-dependent kill switch.

#### *Kill-switches: Cycle 2*

**Design**
We redesigned the methane-dependent kill switch using a [hybrid promoter](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/MethaneKillSwitch) to control toxin production, in which both
methane concentration and cell density serve as input signal [21]. This has the
advantages of an expected increase in the formaldehyde sensitivity of the system, and additionally creates a solution to a problem we encountered during our [human practices](https://2021.igem.org/Team%3AWageningen_UR/Human_Practices) work. If the cows leave
the stable to graze outside, the methane concentration in the biofilter temporarily drops, but the methane-dependent kill switch should not be activated to kill *E. coli*. By using the
hybrid promotor, an “AND gate” is created, so that toxin production is only activated when both methane concentration
and cell density are low. This way, *E. coli* can survive a temporary drop in methane concentration, but is killed when escaping the biofilter, still warranting the safety of the system.

To ensure biocontainment of *P. putida*, we designed a cell density-dependent kill switch. Quorum sensing is a mechanism can be used to ‘sense’ the cell density [22], and will be coupled to the
hok/sok toxin-antitoxin system again. This mechanism relies on the production of the quorum sensing molecule AHL in the cell, which rapidly diffuses in and out of any cell and through the medium.
Therefore, the intracellular AHL concentration will only be high in high cell densities. Toxin production will be placed under the *lux pL* promoter, which is repressed by AHL. This way,
toxin production in repressed in high cell densities (inside the biofilter), and repression is lifted in low cell densities (outside the biofilter), leading to toxin production and cell death.
As the *lux pR* promoter is activated by AHL, antitoxin production is put under control of this promoter so antitoxin is produced in high cell densities.

With the design of these two kill switches and the hybrid promoter, we ensure cell death of both bacteria upon escape of the biofilter.

**Build**
We worked on building both kill switches as genetic circuits in the lab. We replaced the toxin and antitoxin by fluorescent reporters to prevent cloning difficulties and facilitate testing the
dynamics of the systems because fluorescent proteins are easier to measure and do not result in cell death. The proof of concept of the cell density dependent kill switch was built in both
*E. coli* and *P. putida*  and the methane-dependent safety circuit was developed in *E. coli*.

**Test**
We tested the cell density dependent kill switch in the lab using plate reader experiments and a coculture experiment on agar plates and saw a high sensitivity of the circuit to the cell density
(Figure 6). This indicates that quorum sensing is a good input signal to use for the safety circuit. See more details [here](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/QuorumSensing).
Additionally, we tested the toxicity of formaldehyde on strains with a knock-out for the formaldehyde sensing and detoxification pathways. We found out that in wild-type *E. coli*,
of the detoxification enzymes results in a higher toxicity of formaldehyde.

![](https://static.igem.org/mediawiki/2021/f/f3/T--Wageningen_UR--wetlab_QS15.png)
***Figure 6:** AHL sensitivity of the *E. coli* strain containing the reporter-pL-GFP and reporter-pR-RFP plasmids. The sensitivity is calculated by dividing the GFP/RFP ratio without AHL by
the GFP/RFP ratio with AHL.*

**Learn**
We learned that the quorum sensing mechanism is a good input signal to base the cell density dependent kill switch in *P. putida* on, as the circuit was very sensitive to the AHL concentration.

#### *Co-dependency*

**Design**
We have designed our biofilter to harbor two genetically engineered microbes. Although we have designed safety mechanisms for both *E. coli* and **P. putida**, such as the quorum sensing,
we needed to add another layer of protection. We made the bacteria exchange vital nutrients to each other, so that if they are separated outside of the biofilter, both bacteria would die. More
specifically, we engineered *E. coli*. to grow on methane and secrete acetate, while **P. putida** needed to be able to use acetate as its carbon source. In addition, we designed both
microbes to be auxotrophic for one amino acid each. We did this by consulting the available literature and using the iGEM PIPE. Details on how we used this tool for the auxotrophy co-dependency can
be found at our [Auxotrophy design page](https://2021.igem.org/Team%3AWageningen_UR/Software/Auxotrophy_design). ![](https://static.igem.org/mediawiki/2021/c/c5/T--Wageningen_UR--SBD-overview.png)
***Figure 6:** Schematic overview of the three safety mechanisms in E. coli (right) and P. putida (left). 1: methane-dependent kill-switch, 2: proximity-dependent kill-switch, 3: co-dependency.*

**Build**
With this tool we made the microbes auxotrophic and simulated their uptake and production rates for tryptophan (Trp) and arginine (Arg). We also checked that the auxotrophic models of *E. coli* and
**P. putida**were able to grow on methane and acetate respectively.

After designing the strategy using PIPE, we went into the lab. Making knock-ins and knockouts as suggested by The iGEM PIPE in *E. coli* and **P. putida**.

**Test**
The iGEM PIPE predicted that that Arg needed to be supplied in relatively high amount compared to Trp. The simulation also indicated that **P. putida** can consume acetate while secreting
the amino acid needed to complement the auxotrophic *E. coli*. Similarly, *E. coli* could grow on methane while producing acetate and the amino acid complementing **P. putida**’s
auxotrophy.

**Learn/Design**
The main outcome of this application of the iGEM PIPE was that we then knew that Arg was likely not the best amino acid for establishing auxotrophy. This is because one of the two microorganism would
need to produce it in high amount for the other to survive. Nevertheless, we needed to test this experimentally *in vivo* ([See our
Auxotrophy page for more details!](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/Auxotrophy)). Therefore another cycle began!

**Build**
To establish the auxotrophy, we wanted to use amino acids that are only needed by the bacteria in low amounts, so the overproducing bacterium would be able to sustain the receiving bacterium.

To investigate which amino acids are suitable for this, we used the *E. coli* strains from the KEIO collection with knock-outs for specific amino acids. We grew these strains in medium supplemented
with the amino acids to see when wild type growth was restored.

**Test**
The experimental results confirmed the predictions obtained with the iGEM PIPE. For certain amino acids (e.g. Trp and Tyr) low concentrations were sufficient to sustain growth, in contrast with the
high concentrations needed for Arg-auxotrophs. Due to time constraints, we did not get to experimentally prove that the auxotrophic **P. putida** was able to grow on acetate. However,
we could test the relative abundance of the two microorganisms in a co-culture of **P. putida** and *E. coli*. The glucose assimilation pathway was knocked-out in **P. putida**
while *E. coli* was WT. Supplying only glucose as carbon source showed that the co-culture could be maintained by the same 1:1 ratio of the initial culture after 24 hours of growth.

![](https://static.igem.org/mediawiki/2021/9/9e/T--Wageningen_UR--Auxotrophy_graph_science.jpeg)
***Figure 7:** The overproduction of Tyr by *E. coli* and Trp by the *P. putida* was shown to sustain minimal growth of auxotrophic *E. coli* strains for the respective amino acid.
Dashed lines indicate minimal concentration of the amino acid needed for growth.*

**Learn**
In our final design, the amino acid pair of choice was Trp-Tyr, with **P. putida** auxotrophic for Tyr, and *E. coli* for Trp. Additionally, we learned that having a carbon source
co-dependency plays an important role in the maintenance of the ratio between the bacterial strains during co-culture.

Curious for more details on the final design of all our pillars? Visit the [overview page](https://2021.igem.org/Team%3AWageningen_UR/Results)!

### Implementation

We developed Cattlelyst in the lab, but to implement the biofilter, we need to know more: how efficient will Cattlelyst be? What are the physical dimensions of our system? And what are the costs
associated with running such an installation?

To answer these questions and understand the impact Cattlelyst will make, we modelled our biofilter from the microscale, standing for one cell, to the macroscale… the entire biofilter. By doing so,
we could estimate the size and costs as well as show bottlenecks to improve our biofilter. We found out that decreasing the gas flow rate in the biofilter can help make our system even more
efficient in the future. Please refer to the [implementation page](https://2021.igem.org/Team%3AWageningen_UR/Implementation) for more information on the physical design of our biofilter.

* ###### References

  *arrow\_downward*

  1. D. J. Leak and H. Dalton, “Growth yields of methanotrophs - 1. Effect of copper on the energetics of methane oxidation,” Appl. Microbiol. Biotechnol., vol. 23, no. 6, pp. 470–476, 1986, doi: 10.1007/BF02346062.
  2. T. T, G. MT, and H. CA, “Development of a CRISPR/Cas9 System for Methylococcus capsulatus In Vivo Gene Editing,” Appl. Environ. Microbiol., vol. 85, no. 11, Jun. 2019, doi: 10.1128/AEM.00340-19.
  3. F. Y. H. Chen, H. W. Jung, C. Y. Tsuei, and J. C. Liao, “Converting Escherichia coli to a Synthetic Methylotroph Growing Solely on Methanol,” Cell, vol. 182, no. 4, pp. 933-946.e14, Aug. 2020, doi: 10.1016/J.CELL.2020.07.010.
  4. B. Zhao, D. Y. Cheng, P. Tan, Q. An, and J. S. Guo, “Characterization of an aerobic denitrifier Pseudomonas stutzeri strain XL-2 to achieve efficient nitrate removal,” Bioresour. Technol., vol. 250, no. August 2017, pp. 564–573, 2018, doi: 10.1016/j.biortech.2017.11.038.
  5. D. U. Lee, I. S. Lee, Y. D. Choi, and J. H. Bae, “Effects of external carbon source and empty bed contact time on simultaneous heterotrophic and sulfur-utilizing autotrophic denitrification,” Process Biochem., vol. 36, no. 12, pp. 1215–1224, 2001, doi: 10.1016/S0032-9592(01)00163-7.
  6. Q. Y. Zhang, P. Yang, L. S. Liu, and Z. J. Liu, “Formulation and characterization of a heterotrophic nitrification-aerobic denitrification synthetic microbial community and its application to livestock wastewater treatment,” Water (Switzerland), vol. 12, no. 1, 2020, doi: 10.3390/w12010218.
  7. M. Martin-Pascual et al., “A navigation guide of synthetic biology tools for Pseudomonas putida,” 2021, doi: 10.1016/j.biotechadv.2021.107732.
  8. F. Gubellini et al., “Physiological Response to Membrane Protein Overexpression in E. coli,” Mol. Cell. Proteomics, vol. 10, no. 10, Oct. 2011, doi: 10.1074/MCP.M111.007930.
  9. R. K. Bennett et al., “Expression of soluble methane monooxygenase in Escherichia coli enables methane conversion,” bioRxiv, p. 2021.08.05.455234, Aug. 2021, doi: 10.1101/2021.08.05.455234.
  10. S. Pande et al., “Fitness and stability of obligate cross-feeding interactions that emerge upon gene loss in bacteria,” ISME J. 2014 85, vol. 8, no. 5, pp. 953–962, Nov. 2013, doi: 10.1038/ismej.2013.211.
  11. J. M. Wehrfritz, A. Reilly, S. Spiro, and D. J. Richardson, “Purification of hydroxylamine oxidase from Thiosphaera pantotropha. Identification of electron acceptors that couple heterotrophic nitrification to aerobic denitrification,” FEBS Lett., vol. 335, no. 2, pp. 246–250, 1993, doi: 10.1016/0014-5793(93)80739-H.
  12. H. S. Joo, M. Hirai, and M. Shoda, “Characteristics of ammonium removal by heterotrophic nitrification-aerobic denitrification by Alcaligenes faecalis no. 4,” J. Biosci. Bioeng., vol. 100, no. 2, pp. 184–191, 2005, doi: 10.1263/jbb.100.184.
  13. J. Zhang, P. Wu, B. Hao, and Z. Yu, “Heterotrophic nitrification and aerobic denitrification by the bacterium Pseudomonas stutzeri YZN-001,” Bioresour. Technol., vol. 102, no. 21, pp. 9866–9869, Nov. 2011, doi: 10.1016/j.biortech.2011.07.118.
  14. NO is the product of hydroxylamine oxidoreductase
      Jonathan D. Caranto, Kyle M. Lancaster
      Proceedings of the National Academy of Sciences Aug 2017, 114 (31) 8217-8222; DOI: 10.1073/pnas.1704504114
  15. N. Zhang, H. Chen, Y. Lyu, and Y. Wang, “Nitrogen removal by a metal-resistant bacterium, Pseudomonas putida ZN1, capable of heterotrophic nitrification-aerobic denitrification,” 2018, doi: 10.1002/jctb.5863.
  16. O. Yishai, L. Goldbach, H. Tenenboim, S. N. Lindner, and A. Bar-Even, “Engineered Assimilation of Exogenous and Endogenous Formate in Escherichia coli,” ACS Synth. Biol., vol. 6, no. 9, pp. 1722–1731, Sep. 2017, doi: 10.1021/ACSSYNBIO.7B00086.
  17. C. A. Henard, H. Smith, N. Dowe, M. G. Kalyuzhnaya, P. T. Pienkos, and M. T. Guarnieri, “Bioconversion of methane to lactate by an obligate methanotrophic bacterium,” Sci. Reports 2016 61, vol. 6, no. 1, pp. 1–9, Feb. 2016, doi: 10.1038/srep21585.
  18. H. J. Kim et al., “Biological conversion of methane to methanol through genetic reassembly of native catalytic domains,” Nat. Catal. 2019 24, vol. 2, no. 4, pp. 342–353, Apr. 2019, doi: 10.1038/s41929-019-0255-1.
  19. D. Osman et al., “The Effectors and Sensory Sites of Formaldehyde-responsive Regulator FrmR and Metal-sensing Variant \* □,” vol. 291, no. 37, pp. 19502–19516, 2016, doi: 10.1074/jbc.M116.745174.
  20. K. Gerdes, L. K. Poulsen, T. Thisted, A. K. Nielsen, J. Martinussen, and P. H. Andreasen, “The hok killer gene family in gram-negative bacteria,” vol. 2, no. 11, pp. 946–956, 1990.
  21. M. B. Miller et al., “Tuning the dynamic range of bacterial promoters regulated by ligand-inducible transcription factors,” Nat. Commun., vol. 9, no. 1, pp. 1–8, 2018, doi: 10.1038/s41467-017-02473-5.
  22. M. B. Miller and B. L. Bassler, “Quorum Sensing in Bacteria,” Annu. Rev. Microbiol., vol. 5, no. 1, pp. 165–199, 2001.

##### About Cattlelyst

Cattlelyst is the name of the iGEM 2021 WUR team. Our name is a mix of 1) our loyal furry friends, cattle, and 2) catalyst, which is something that increases the rate of a reaction.
We are developing “the something” that converts the detrimental gaseous emissions of cattle, hence our name Cattlelyst.

Are you curious about our journey? We have written about our adventures in our blog, which you can find here:

##### Keep in touch!

* igem@wur.nl
* [iGEMWageningen](https://www.facebook.com/iGEMWageningen/?hc_ref=ARTIdtcStpEkeL1jRvNQs5XrYIZk9-Nl-Ieh6o3DXz7A9PsWjnip9gC1W0BEGBf_9Rg&fref=nf&__tn__=kC-R)
* [@igemwageningen](https://twitter.com/igemwageningen?ref_src=twsrc%5Egoogle%7Ctwcamp%5Eserp%7Ctwgr%5Eauthor)
* [iGEM Wageningen](https://nl.linkedin.com/company/igem-wageningen)
* [igem.wageningen](https://www.instagram.com/igem.wageningen/)

![logo](https://static.igem.org/mediawiki/2021/f/f0/T--Wageningen_UR--Cattlelyst_logo.png "Logo")

*format\_list\_bulleted*

* ###### SECTIONS
* [*arrow\_upward*](#home)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Wageningen UR/Experiments

iGEM Wageningen 2021

[![logo](https://static.igem.org/mediawiki/2021/f/f0/T--Wageningen_UR--Cattlelyst_logo.png "Logo")](https://2021.igem.org/Team%3AWageningen_UR)
*menu*

* [![logo](https://static.igem.org/mediawiki/2021/2/27/T--Wageningen_UR--Cattlelyst_logo_small.png "Logo")](https://2021.igem.org/Team%3AWageningen_UR)
* Project*arrow\_drop\_down*

+ [Description](https://2021.igem.org/Team%3AWageningen_UR/Description)
+ [Engineering](https://2021.igem.org/Team%3AWageningen_UR/Engineering)
+ [Background](background-final.html)
+ [Sustainable development goals](https://2021.igem.org/Team%3AWageningen_UR/Sustainable)
+ [Contribution](https://2021.igem.org/Team%3AWageningen_UR/Contribution)

* Results*arrow\_drop\_down*

+ [Overview](https://2021.igem.org/Team%3AWageningen_UR/Results)
+ [Modeling](https://2021.igem.org/Team%3AWageningen_UR/Model)
+ [Wetlab](wetlab.html)
+ [Achievements](achievements.html)
+ [Notebook](https://2021.igem.org/Team%3AWageningen_UR/Notebook)

* Bioparts*arrow\_drop\_down*

+ [Pipeline](https://2021.igem.org/Team%3AWageningen_UR/Software)
+ [Wikibase](wikibase.html)
+ [Biobricks](https://2021.igem.org/Team%3AWageningen_UR/Parts)

* Human Practices*arrow\_drop\_down*

+ [Integrated Human Practices](https://2021.igem.org/Team%3AWageningen_UR/Human_Practices)
+ [Communication](https://2021.igem.org/Team%3AWageningen_UR/Communication)
+ [Education](https://2021.igem.org/Team%3AWageningen_UR/Education)
+ [Collaborations](https://2021.igem.org/Team%3AWageningen_UR/Collaborations)
+ [Implementation](https://2021.igem.org/Team%3AWageningen_UR/Implementation)

* Safety*arrow\_drop\_down*

+ [Safety by design](safetyByDesign.html)
+ [Lab safety](https://2021.igem.org/Team%3AWageningen_UR/Safety)
+ [Ethics](ethics.html)

* About us*arrow\_drop\_down*

+ [Team](teams.html)
+ [Our journey](ourJourney.html)
+ [Attributions](https://2021.igem.org/Team%3AWageningen_UR/Attributions)
+ [Sponsors](sponsors.html)

* [![logo](https://static.igem.org/mediawiki/2021/f/f0/T--Wageningen_UR--Cattlelyst_logo.png "Logo")](https://2021.igem.org/Team%3AWageningen_UR)
  [*clear*](#!)
* + Project*arrow\_drop\_down*

    - [Description](https://2021.igem.org/Team%3AWageningen_UR/Description)
    - [Engineering](https://2021.igem.org/Team%3AWageningen_UR/Engineering)
    - [Background](background-final.html)
    - [Sustainable development goals](https://2021.igem.org/Team%3AWageningen_UR/Sustainable)
    - [Contribution](https://2021.igem.org/Team%3AWageningen_UR/Contribution)
  + Results*arrow\_drop\_down*

    - [Overview](https://2021.igem.org/Team%3AWageningen_UR/Results)
    - [Modeling](https://2021.igem.org/Team%3AWageningen_UR/Model)
    - [Wetlab](wetlab.html)
    - [Achievements](achievements.html)
    - [Notebook](https://2021.igem.org/Team%3AWageningen_UR/Notebook)
  + Bioparts*arrow\_drop\_down*

    - [Pipeline](https://2021.igem.org/Team%3AWageningen_UR/Software)
    - [Wikibase](wikibase.html)
    - [Biobricks](https://2021.igem.org/Team%3AWageningen_UR/Parts)
  + Human Practices*arrow\_drop\_down*

    - [Integrated Human Practices](https://2021.igem.org/Team%3AWageningen_UR/Human_Practices)
    - [Communication](https://2021.igem.org/Team%3AWageningen_UR/Communication)
    - [Education](https://2021.igem.org/Team%3AWageningen_UR/Education)
    - [Collaborations](https://2021.igem.org/Team%3AWageningen_UR/Collaborations)
    - [Implementation](https://2021.igem.org/Team%3AWageningen_UR/Implementation)
  + Safety*arrow\_drop\_down*

    - [Safety by design](safetyByDesign.html)
    - [Lab safety](https://2021.igem.org/Team%3AWageningen_UR/Safety)
    - [Ethics](ethics.html)
  + About us*arrow\_drop\_down*

    - [Team](teams.html)
    - [Our journey](ourJourney.html)
    - [Attributions](https://2021.igem.org/Team%3AWageningen_UR/Attributions)
    - [Sponsors](sponsors.html)

# Experiments

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).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [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)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Wageningen UR/Results

iGEM Wageningen 2021

[![logo](https://static.igem.org/mediawiki/2021/f/f0/T--Wageningen_UR--Cattlelyst_logo.png "Logo")](https://2021.igem.org/Team%3AWageningen_UR)
*menu*

* [![logo](https://static.igem.org/mediawiki/2021/2/27/T--Wageningen_UR--Cattlelyst_logo_small.png "Logo")](https://2021.igem.org/Team%3AWageningen_UR)
* Project*arrow\_drop\_down*

+ [Description](https://2021.igem.org/Team%3AWageningen_UR/Description)
+ [Engineering](https://2021.igem.org/Team%3AWageningen_UR/Engineering)
+ [Background](https://2021.igem.org/Team%3AWageningen_UR/Background)
+ [Implementation](https://2021.igem.org/Team%3AWageningen_UR/Implementation)
+ [Sustainable development goals](https://2021.igem.org/Team%3AWageningen_UR/Sustainable)
+ [Contribution](https://2021.igem.org/Team%3AWageningen_UR/Contribution)

* Results*arrow\_drop\_down*

+ [Overview](https://2021.igem.org/Team%3AWageningen_UR/Results)
+ [Modeling](https://2021.igem.org/Team%3AWageningen_UR/Model)
+ [Wetlab](https://2021.igem.org/Team%3AWageningen_UR/Wetlab)
+ [Achievements](https://2021.igem.org/Team%3AWageningen_UR/Achievements)
+ [Notebook](https://2021.igem.org/Team%3AWageningen_UR/Notebook)

* Bioparts*arrow\_drop\_down*

+ [The iGEM PIPE](https://2021.igem.org/Team%3AWageningen_UR/Software)
+ [Wikibase](https://2021.igem.org/Team%3AWageningen_UR/Wikibase)
+ [Biobricks](https://2021.igem.org/Team%3AWageningen_UR/Parts)

* Human Practices*arrow\_drop\_down*

+ [Integrated Human Practices](https://2021.igem.org/Team%3AWageningen_UR/Human_Practices)
+ [Communication](https://2021.igem.org/Team%3AWageningen_UR/Communication)
+ [Education](https://2021.igem.org/Team%3AWageningen_UR/Education)
+ [Collaborations](https://2021.igem.org/Team%3AWageningen_UR/Collaborations)
+ [Dutch Meetup](https://2021.igem.org/Team%3AWageningen_UR/DutchMeetup)
+ [Lab Olympics](https://2021.igem.org/Team%3AWageningen_UR/LabOlympics)

* Safety*arrow\_drop\_down*

+ [Safety by design](https://2021.igem.org/Team%3AWageningen_UR/SafetyByDesign)
+ [Lab safety](https://2021.igem.org/Team%3AWageningen_UR/Safety)
+ [Ethics](https://2021.igem.org/Team%3AWageningen_UR/Ethics)

* About us*arrow\_drop\_down*

+ [Team](https://2021.igem.org/Team%3AWageningen_UR/Team)
+ [Our journey](https://2021.igem.org/Team%3AWageningen_UR/OurJourney)
+ [Attributions](https://2021.igem.org/Team%3AWageningen_UR/Attributions)
+ [Sponsors](https://2021.igem.org/Team%3AWageningen_UR/Sponsors)

* [![logo](https://static.igem.org/mediawiki/2021/f/f0/T--Wageningen_UR--Cattlelyst_logo.png "Logo")](https://2021.igem.org/Team%3AWageningen_UR)
  [*clear*](#!)
* + Project*arrow\_drop\_down*

    - [Description](https://2021.igem.org/Team%3AWageningen_UR/Description)
    - [Engineering](https://2021.igem.org/Team%3AWageningen_UR/Engineering)
    - [Background](https://2021.igem.org/Team%3AWageningen_UR/Background)
    - [Sustainable development goals](https://2021.igem.org/Team%3AWageningen_UR/Sustainable)
    - [Contribution](https://2021.igem.org/Team%3AWageningen_UR/Contribution)
    - [Implementation](https://2021.igem.org/Team%3AWageningen_UR/Implementation)
  + Results*arrow\_drop\_down*

    - [Overview](https://2021.igem.org/Team%3AWageningen_UR/Results)
    - [Modeling](https://2021.igem.org/Team%3AWageningen_UR/Model)
    - [Wetlab](https://2021.igem.org/Team%3AWageningen_UR/Wetlab)
    - [Achievements](https://2021.igem.org/Team%3AWageningen_UR/Achievements)
    - [Notebook](https://2021.igem.org/Team%3AWageningen_UR/Notebook)
  + Bioparts*arrow\_drop\_down*

    - [The iGEM PIPE](https://2021.igem.org/Team%3AWageningen_UR/Software)
    - [Wikibase](https://2021.igem.org/Team%3AWageningen_UR/Wikibase)
    - [Biobricks](https://2021.igem.org/Team%3AWageningen_UR/Parts)
  + Human Practices*arrow\_drop\_down*

    - [Integrated Human Practices](https://2021.igem.org/Team%3AWageningen_UR/Human_Practices)
    - [Communication](https://2021.igem.org/Team%3AWageningen_UR/Communication)
    - [Education](https://2021.igem.org/Team%3AWageningen_UR/Education)
    - [Collaborations](https://2021.igem.org/Team%3AWageningen_UR/Collaborations)
    - [Dutch Meetup](https://2021.igem.org/Team%3AWageningen_UR/DutchMeetup)
    - [Lab Olympics](https://2021.igem.org/Team%3AWageningen_UR/LabOlympics)
  + Safety*arrow\_drop\_down*

    - [Safety by design](https://2021.igem.org/Team%3AWageningen_UR/SafetyByDesign)
    - [Lab safety](https://2021.igem.org/Team%3AWageningen_UR/Safety)
    - [Ethics](https://2021.igem.org/Team%3AWageningen_UR/Ethics)
  + About us*arrow\_drop\_down*

    - [Team](https://2021.igem.org/Team%3AWageningen_UR/Team)
    - [Our journey](https://2021.igem.org/Team%3AWageningen_UR/OurJourney)
    - [Attributions](https://2021.igem.org/Team%3AWageningen_UR/Attributions)
    - [Sponsors](https://2021.igem.org/Team%3AWageningen_UR/Sponsors)

# Overview

![Engineering Photo](https://static.igem.org/mediawiki/2021/0/09/T--Wageningen_UR--Parallax_overview.jpg)

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](https://2021.igem.org/Team%3AWageningen_UR/Wetlab) or via hyperlinks within this page.

![Icon ammonia](https://static.igem.org/mediawiki/2021/5/50/T--Wageningen_UR--Icon_ammonia.png)

### Ammonia conversion

![Icon ammonia](https://static.igem.org/mediawiki/2021/5/50/T--Wageningen_UR--Icon_ammonia.png)

### Ammonia conversion

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](https://2021.igem.org/Team%3AWageningen_UR/Model/Nitrogen) to increase our conceptual understanding of HNAD. This process is divided in two different processes, namely [nitrification](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/Nitrification) and [denitrification](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/SyntheticDenitrification). 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](https://2021.igem.org/Team%3AWageningen_UR/Engineering).

![Icon ammonia](https://static.igem.org/mediawiki/2021/7/7a/T--Wageningen_UR--nitrogen-path.png)

**Figure 1.** Illustration of the pathway for the conversion of ammonia. The orange boxes indicate the enzymes. AMO = ammonia monooxygenase, HAO = Hydroxylamine oxidoreductase,
Nap = periplasmic nitrate reductase, Nir = nitrite reductase, Nor = nitric oxide reductase, Nos = nitrous oxide reductase.

#### Nitrification

Although the exact mechanisms or pathway for nitrification are not known, the conversion of NH3 to hydroxylamine (NH2OH) and subsequently to NO2- or
NO facilitated by ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO), respectively [6], [7]. To date, the genomic sequences of AMO and HAO from heterotrophic nitrifiers have
never been fully characterized [8]. Therefore, we used the genes from the well characterized autotrophic nitrifier *Nitrosomonas europaea*.

Two other iGEM teams have attempted to heterologously express the *N. europaea* nitrification genes. The [Virginia 2017 team](https://2017.igem.org/Team%3AVirginia) tried to express both
AMO and HAO with the corresponding cytochromes (c554 and cycX) from *N. europaea* in *Paracoccus denitrificans*. However, they only succeeded in expressing HAO properly. The [DTU Denmark 2013 team](https://2013.igem.org/Team%3ADTU-Denmark)
tried as well, but only succeeded in expressing AMO in *Escherichia coli* (*E. coli*). Neither team used codon harmonization. Therefore, we decided to apply codon harmonization in our
chassis *P. putida* for both AMO and HAO, and their corresponding cytochromes (c554, c552 and cycX). The different subunits or genes of AMO or HAO were combined on a plasmid into one
operon with a synthetic ribosome binding site (RBS) between each subunit. All plasmids were successfully cloned into *P. putida* (Figure 2).

![](https://static.igem.org/mediawiki/2021/5/52/T--Wageningen_UR--Overview_colPCR_Thomas.png)

**Figure 2.** Gel electrophoresis analysis of a colony PCR. *Pseudomonas putida* EM42 was checked for the presence of plasmids with inserts containing nitrification genes from *Nitrosomonas europaea*.
pCX = plasmid with accessory genes cytochrome c554 and cycX; pAMO = plasmid with ammonia monooxygenase genes *amoC*, *amoA*, and *amoB*; pHAO = plasmid with hydroxylamine oxidoreductase genes *haoA* and *haoB*;
numbers indicate the expected PCR product size in base pairs. Band height matches the expected product size, confirming that this *P. putida* strain
harbors all three plasmids.

Although the exact mechanisms or pathway for nitrification are not known, the conversion of NH3 to hydroxylamine (NH2OH) and subsequently to NO2- or
NO facilitated by ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO), respectively [6], [7]. To date, the genomic sequences of AMO and HAO from heterotrophic nitrifiers have
never been fully characterized [8]. Therefore, we used the genes from the well characterized autotrophic nitrifier *Nitrosomonas europaea*.

Two other iGEM teams have attempted to heterologously express the *N. europaea* nitrification genes. The [Virginia 2017 team](https://2017.igem.org/Team%3AVirginia) tried to express both
AMO and HAO with the corresponding cytochromes (c554 and cycX) from *N. europaea* in *Paracoccus denitrificans*. However, they only succeeded in expressing HAO properly. The [DTU Denmark 2013 team](https://2013.igem.org/Team%3ADTU-Denmark)
tried as well, but only succeeded in expressing AMO in *Escherichia coli* (*E. coli*). Neither team used codon harmonization. Therefore, we decided to apply codon harmonization for our
chassis *P. putida* for both AMO and HAO, and their corresponding cytochromes (c554, c552 and cycX). The different subunits or genes of AMO or HAO were combined on a plasmid into one
operon with a synthetic ribosome binding site (RBS) between each subunit. All plasmids were successfully cloned into *P. putida* (Figure 2).

![](https://static.igem.org/mediawiki/2021/5/52/T--Wageningen_UR--Overview_colPCR_Thomas.png)

**Figure 2.** Gel electrophoresis analysis of a colony PCR. *Pseudomonas putida* EM42 was checked for the presence of plasmids with inserts containing nitrification genes from *Nitrosomonas europaea*.
pCX = plasmid with accessory genes cytochrome c554 and cycX; pAMO = plasmid with ammonia monooxygenase genes *amoC*, *amoA*, and *amoB*; pHAO = plasmid with hydroxylamine oxidoreductase genes *haoA* and *haoB*;
numbers indicate the expected PCR product size in base pairs. Band height matches the expected product size, confirming that this *P. putida* strain
harbors all three plasmids.

[![](https://static.igem.org/mediawiki/2021/0/04/T--Wageningen_UR--Icon_nitrification.png)](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/Nitrification)

###### Nitrification

#### 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.

In contrast to nitrification, we could use the genes from known aerobic denitrifiers, as their denitrification genes are characterized. Enzymes from the following known aerobic denitrifiers were tested:
*Paracoccus denitrificans* and *Pseudomonas stutzeri* [9]–[12]. Additionally, the genes from the denitrifier *Cupriavidus necator* were used. This bacterium
is not known for aerobic denitrification, but it does possess Nap, which is specifically known for being active in aerobic conditions [11]. By using the genes from different organisms, multiple variants
of the enzymes could be tested.

Within the timeframe of the iGEM competition, we were able to successfully express the Nap and Nir enzymes in one strain. This strain was grown with NO3- for 24 hours (Figure 2).
Clearly, more NO2- is produced with Nap alone in comparison to Nap + Nir. This indicates that the produced NO2- from Nap is consecutively consumed by Nir.
These results indicate that the enzymes are simultaneously functional (Figure 3).

![](https://static.igem.org/mediawiki/2021/9/9b/T--Wageningen_UR--Wetlab_synDenit_napNir.png)

**Figure 3.** NO2- production by *P. putida* EM42 ∆nasT containing the nap operon originating from **P. denitrificans** on plasmid
(Nap) or containing the nir operon origination from *P. stutzeri* on plasmid as well (Nap + Nir). The NO2- production was corrected for the change in OD over the
time span of 24 hours. As control, *P. putida* EM42 ∆nasT containing pSEVAb22 without cargo was used. Three biological replicates were used for the control and five biological replicates
for Nap and Nap + Nir.

In contrast to nitrification, we could use the genes from known aerobic denitrifiers, as their denitrification genes are characterized. Enzymes from the following known aerobic denitrifiers were tested:
*Paracoccus denitrificans* and *Pseudomonas stutzeri* [9]–[12]. Additionally, the genes from the denitrifier *Cupriavidus necator* were used. This bacterium
is not known for aerobic denitrification, but it does possess Nap, which is specifically known for being active in aerobic conditions [11]. By using the genes from different organisms, multiple variants
of the enzymes could be tested.

Within the timeframe of the iGEM competition, we were able to successfully express the Nap and Nir enzymes in one strain. This strain was grown with NO3- for 24 hours (Figure 2).
Clearly, more NO2- is produced with Nap alone in comparison to Nap + Nir. This indicates that the produced NO2- from Nap is consecutively consumed by Nir.
These results indicate that the enzymes are simultaneously functional (Figure 3).

![](https://static.igem.org/mediawiki/2021/9/9b/T--Wageningen_UR--Wetlab_synDenit_napNir.png)

**Figure 3.** NO2- production by *P. putida* EM42 ∆nasT containing the nap operon originating from **P. denitrificans** on plasmid
(Nap) or containing the nir operon origination from *P. stutzeri* on plasmid as well (Nap + Nir). The NO2- production was corrected for the change in OD over the
time span of 24 hours. As control, *P. putida* EM42 ∆nasT containing pSEVAb22 without cargo was used. Three biological replicates were used for the control and five biological replicates
for Nap and Nap + Nir.

[![](https://static.igem.org/mediawiki/2021/e/e1/T--Wageningen_UR--Icon_denitrification.png)](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/SyntheticDenitrification)

###### Denitrification - Mosaic approach

##### 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.

![](https://static.igem.org/mediawiki/2021/4/4e/T--Wageningen_UR--Wetlab_Sanne_figure_12.png)

**Figure 4.**  NO2- production by *P. putida* EM42 ∆nasT containing the nap operon originating from **P. denitrificans** on plasmid
(Nap plasmid) and *P. putida* :SD grown without IPTG (:SD) and with IPTG (:SD+). The NO2- production was corrected for the change in OD over the time span of 24 hours.
As control, *P. putida* EM42 ∆nasT was used. Three biological replicates were used for the control, :SD and :SD+ and five biological replicates for Nap plasmid.

Given that we placed the denitrification pathway under the control of 2 T7 promoters, we needed to integrate a T7 polymerase (T7pol). This polymerase specifically transcribes DNA only downstream
of a T7 promoter, synthesizes RNA at a high rate. Additionally, the T7pol ignores terminators, which increases the likelihood that the whole pathway is transcribed [14]. Moreover, we made T7-polymerase
expression IPTG inducible. This was done to prevent constitutive expression, and with that the associated metabolic burden of the denitrification cassette. The final strain was called *P. putida* :SD, SD standing for Synthetic Denitrification.

Within the timeframe of the iGEM competition, we were able to test NO2- and N2O accumulation. For NO2- accumulation specifically, the strains
were grown with NO3- for 24 hours. Furthermore, we tested the effect of IPTG on nitrogen dynamics. The strains were grown with with IPTG (:SD+) and without IPTG (:SD). For the
:SD+ condition, the pathway is transcribed to a higher extend. Compared to the control, more NO2- accumulates for :SD and :SD+, suggesting that the NO3-
reductase works. However, when compared with the Nap plasmid, less NO2- accumulates (Figure 4). This could imply two things: (1) nitrate reduction is not as optimal as for the plasmid or
(2) the coupled NO2- reductase works too. The fact that the :SD with IPTG accumulates less NO2- compared to the :SD suggests that more
NO2- is reduced. This could be explained by higher expression rates downstream of the pathway.

Given that we placed the denitrification pathway under the control of 2 T7 promoters, we needed to integrate a T7 polymerase (T7pol). This polymerase specifically transcribes DNA only downstream
of a T7 promoter, synthesizes RNA at a high rate. Additionally, the T7pol ignores terminators which increases the likelihood tha the whole pathway is transcribed [14]. Moreover, we made T7-polymerase
expression IPTG inducible. This was done to prevent constitutive expression, and with that the associated metabolic burden of the denitrification cassette. The final strain was called *P. putida*
:SD, SD standing for Synthetic Denitrification.

![](https://static.igem.org/mediawiki/2021/4/4e/T--Wageningen_UR--Wetlab_Sanne_figure_12.png)

**Figure 4.** NO2- production by *P. putida* EM42 ∆nasT containing the nap operon originating from **P. denitrificans** on plasmid
(Nap plasmid) and *P. putida* :SD growth without IPTG (:SD) and with IPTG (:SD+) . The NO2- production was corrected for the change in OD over the time span of 24 hours.
As control, *P. putida* EM42 ∆nasT was used. Three biological replicates were used for the control, :SD and :SD+ and five biological replicates for Nap plasmid.

Within the timeframe of the iGEM competition, we were able to test NO2- and N2O accumulation. For NO2- accumulation specifically, the strains
were grown with NO3- for 24 hours. Furthermore, we tested the effect of IPTG on nitrogen dynamics, the strains were grown with with IPTG (:SD+) and without IPTG (:SD). For the
:SD+ condition, the pathway is transcribed to a higher extend. Compared to the control, more NO2- accumulates for :SD and :SD+, suggesting that the NO3-
reductase works. However, when compared with the Nap plasmid, less NO2- accumulates (Figure 4). This could imply two things: (1) nitrate reduction is not as optimal as for the plasmid or
(2) the coupled NO2- reductase works too. The fact that the :SD with IPTG accumulates less NO2- compared to the :SD suggests that more
NO2- is reduced. This could be explained by higher expression rates downstream of the pathway.

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.

[![](https://static.igem.org/mediawiki/2021/a/ae/T--Wageningen_UR--Icon_nitrous.png)](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/ElectronBalance)

###### Denitrification - Plug-and-play approach

##### 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.

![](https://static.igem.org/mediawiki/2021/9/9a/T--Wageningen_UR--Wetlab_Sanne_figure_9.png)

**Figure 5.** 16-hour growth curves of *P. putida* EM42 with the terminal oxidases downregulated. Each line represents the downregulation effect of a spacers targeting
different terminal oxidases. Values represent the mean and standard deviation of (1) doubling time, (2) start of exponential phase, (3) biomass yield for three biological and two technical replicates.
Note that for spacer 4, 11, 15, only two biological replicates were tested.

[![](https://static.igem.org/mediawiki/2021/a/ae/T--Wageningen_UR--Icon_nitrous.png)](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/ElectronBalance)

###### Denitrification - Limit N2O

![Icon ammonia](https://static.igem.org/mediawiki/2021/2/22/T--Wageningen_UR--Icon_methane.png)

### Methane Oxidation

![Icon ammonia](https://static.igem.org/mediawiki/2021/2/22/T--Wageningen_UR--Icon_methane.png)

### Methane Oxidation

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](https://2021.igem.org/Team%3AWageningen_UR/Engineering). 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](https://2021.igem.org/Team%3AWageningen_UR/Software). 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](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/MMO) and [Engineering cycle page](https://2021.igem.org/Team%3AWageningen_UR/Engineering).
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.

![Icon ammonia](https://static.igem.org/mediawiki/2021/1/1a/T--Wageningen_UR--methane-path-sanne.png)

**Figure 6.** Methane conversion pathway. Methane is converted to methanol via soluble or particulate methane monooxygenase (sMMO/pMMO). Methanol is subsequently converted to formaldehyde via
methanoldehydrogenase (Mdh). Formaldehyde/formate can be incorporated into biomass or converted into carbon dioxide.

#### 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](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/MMO). 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

Two types of MMO have been engineered within this project. First for the sMMO variant, a similar approach to both the *E. Cowli* strain from [iGEM Braunschweig 2014](https://2014.igem.org/Team%3ABraunschweig/Project) and the Bennet et al. 2021 paper
is used [17]. Here the entire operon from the  *M. capsulatus*  species is taken and expressed together with chaperone proteins. Unfortunately, this aspect never got out of the cloning phase and thus
this project was never tested. The second approach is the heterologous expression of the pMMO, for which the method of Kim et al. 2019 was used [18]. This proved more fruitful as the simpler cloning steps
allowed us to test this enzyme both *in vivo* and *in vitro*. Using Sonification, filtration and His-Tag purification of the product the presence of the enzyme was suggested on SDS-page as can be
seen in Figure 7. No enzyme activity was detected after 25 hours of incubation of the purified enzyme in a methane rich atmosphere. After iGEM this project will continue and other mimics of pMMO will be
expressed and tested in  *in vitro* . Additionally, sMMO will be expressed in one of the C1 growing strain, engineering a synthetic methanotroph.

![](https://static.igem.org/mediawiki/2021/f/fb/T--Wageningen_UR--MMO_figure_7.png)

**Figure 7.** SDS-page of particulate methane monooxygenase and scaffold. pMMO mimic as described in Kim et al 2019, expressed in **E. coli**  BL21 strain with
pET-28(+)-pMMO-m1 plasmid. The lanes harbor left to right (1) PageRuler Prestained Protein Ladder product #26616. (2) Product after Sonication. (3) Product after filtered. (4) Product after
His-tag purification. The expected product size is 54 kDA in all cases and is annotated with the white asterisk in the figure.

Two types of MMO have been engineered within this project. First for the sMMO variant, a similar approach to both the *E. Cowli* strain from iGEM Braunschweig 2014 and the Bennet et al. 2021 paper
is used [17]. Here the entire operon from the  *M. capsulatus*  species is taken and expressed together with chaperone proteins. Unfortunately, this aspect never got out of the cloning phase and thus
this project was never tested. The second approach is the heterologous expression of the pMMO, for which the method of Kim et al. 2019 was used [18]. This proved more fruitful as the simpler cloning steps
allowed us to test this enzyme both *in vivo* and *in vitro*. Using Sonification, filtration and His-Tag purification of the product the presence of the enzyme was suggested on SDS-page as can be
seen in Figure 7. No enzyme activity was detected after 25 hours of incubation of the purified enzyme in a methane rich atmosphere. After iGEM this project will continue and other mimics of pMMO will be
expressed and tested in  *in vitro* . Additionally, sMMO will be expressed in one of the C1 growing strain, engineering a synthetic methanotroph.

![](https://static.igem.org/mediawiki/2021/f/fb/T--Wageningen_UR--MMO_figure_7.png)

**Figure 7.** SDS-page of particulate methane monooxygenase and scaffold. pMMO mimic as described in Kim et al 2019, expressed in **E. coli**  BL21 strain with
pET-28(+)-pMMO-m1 plasmid. The lanes harbor left to right (1) PageRuler Prestained Protein Ladder product #26616. (2) Product after Sonication. (3) Product after filtered. (4) Product after
His-tag purification. The expected product size is 54 kDA in all cases and is annotated with the white asterisk in the figure.

[![](https://static.igem.org/mediawiki/2021/e/e2/T--Wageningen_UR--Icon_methaneConvers.png)](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/MMO)

###### Methane oxidation

![Icon safety](https://static.igem.org/mediawiki/2021/7/7e/T--Wageningen_UR--Icon_safety.png)

### Safety

![Icon safety](https://static.igem.org/mediawiki/2021/7/7e/T--Wageningen_UR--Icon_safety.png)

### Safety

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 P*frm* [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](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/MethaneKillSwitch) 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](https://2021.igem.org/Team%3AWageningen_UR/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.

[![](https://static.igem.org/mediawiki/2021/5/5e/T--Wageningen_UR--Icon_methaneKill.png)](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/MethaneKillSwitch)

###### Methane-dependent kill switch

#### Proximity-dependent kill switch

The second of our safety mechanisms is the proximity-dependent kill switch. The cell density can be ‘sensed’ by using quorum sensing. Quorum sensing is the ability of microorganisms to
regulate gene expression upon different cell density by exchanging signaling molecules [24]. In *Vibrio fischeri* the quorum-sensing molecule (AHL) is produced by the LuxI ([BBa\_C0061](http://parts.igem.org/Part%3ABBa_C0061))
protein. This molecule binds to LuxR, which can either activate the lux pR promoter
([BBa\_R0062](http://parts.igem.org/Part%3ABBa_R0062)) or repress the lux pL promoter ([BBa\_R0063](http://parts.igem.org/Part%3ABBa_R0063%3AExperience))
[25], [26]. In our envisioned circuit, a
toxin is put under control of the *lux pL* promoter and the corresponding antitoxin under control of the *lux pR* promoter as explained on the [proximity based kill switch page](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/QuorumSensing).
We have shown that AHL rich medium is able to both activate the *lux pR* promoter and repress the *lux pL* promoter when they are put together in our reporter strain (see *Figure 17*).
These results show that this safety circuit could ensure biocontainment of the microorganisms as it makes them dependent on the high cell density in the biofilter.

![](https://static.igem.org/mediawiki/2021/e/ec/T--Wageningen_UR--wetlab_QS13.png)

**Figure 8.** Fluorescence of the reporter-pL-GFP and reporter-pR-RFP strain after growing for 11 hours in 25% AHL rich medium (blue), simulating high cell density, or
without AHL (green) medium, simulating a low cell density. The fluorescence is corrected by the OD and normalized for the fluorescence where 0% AHL medium is added.

The second of our safety mechanisms is the cell density-based safety circuit . The cell density can by ‘sensed’ by using quorum sensing. Quorum sensing is the ability of microorganisms to
regulate gene expression upon different cell density by exchanging signaling molecules [24]. In *Vibrio fischeri* the quorum-sensing molecule (AHL) is produced by the LuxI ([BBa\_C0061](http://parts.igem.org/Part%3ABBa_C0061))
protein. This molecule binds to LuxR ([BBa\_C0062](http://parts.igem.org/Part%3ABBa_C0062) which can either activate the lux pR promoter
([BBa\_R0062](http://parts.igem.org/Part%3ABBa_R0062)) or repress the lux pL promoter ([BBa\_R0063](http://parts.igem.org/Part%3ABBa_R0063%3AExperience))
[25], [26]. In our envisioned circuit, a
toxin is put under control of the *lux pL* promoter and the corresponding antitoxin under control of the *lux pR* promoter as explained on the [proximity based kill switch page](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/QuorumSensing).
We have shown that AHL rich medium is able to both activate the *lux pR* promoter and repress the *lux pL* promoter when they are put together in our reporter strain (see *Figure 17*).
These results show that this safety circuit could ensure biocontainment of the microorganisms as it makes them dependent on the high cell density in the biofilter.

![](https://static.igem.org/mediawiki/2021/e/ec/T--Wageningen_UR--wetlab_QS13.png)

**Figure 8.** Fluorescence of the reporter-pL-GFP and reporter-pR-RFP strain after growing for 11 hours in 25% AHL rich medium (blue), simulating high cell density, or
without AHL (green) medium, simulating a low cell density. The fluorescence is corrected by the OD and normalized for the fluorescence where 0% AHL medium is added.

![](https://static.igem.org/mediawiki/2021/f/f3/T--Wageningen_UR--wetlab_QS15.png)

**Figure 9.** AHL sensitivity of the **E. coli** strain containing the reporter-pL-GFP and reporter-pR-RFP plasmids. The sensitivity is calculated by dividing the
GFP/RFP ratio without AHL by the GFP/RFP ratio with AHL.

For the functioning of the biosafety circuit, the toxin/antitoxin ratio in high cell densities versus low cell densities is important [27]. To obtain a sufficient big change in the toxin/antitoxin ratio
in high versus low cell densities, the circuit needs to be sensitive to an input signal, in this case AHL concentration. The sensitivity of the circuit to the AHL concentration (with is related to the
cell density) is calculated by:

![](https://static.igem.org/mediawiki/2021/b/be/T--Wageningen_UR--wetlab_QS14.png)

As can be seen in *Figure 9*, the circuit is sensitive to the cell density and the sensitivity increases over time.

For the functioning of the biosafety circuit, the toxin/antitoxin ratio in high cell densities versus low cell densities is important [27]. To obtain a sufficient big change in the toxin/antitoxin ratio
in high versus low cell densities, the circuit needs to be sensitive to an input signal, in this case AHL concentration. The sensitivity of the circuit to the AHL concentration (with is related to the
cell density) is calculated by:

![](https://static.igem.org/mediawiki/2021/b/be/T--Wageningen_UR--wetlab_QS14.png)

As can be seen in *Figure 9*, the circuit is sensitive to the cell density and the sensitivity increases over time.

![](https://static.igem.org/mediawiki/2021/f/f3/T--Wageningen_UR--wetlab_QS15.png)

**Figure 9.** AHL sensitivity of the **E. coli** strain containing the reporter-pL-GFP and reporter-pR-RFP plasmids. The sensitivity is calculated by dividing the
GFP/RFP ratio without AHL by the GFP/RFP ratio with AHL.

##### 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.

![](https://static.igem.org/mediawiki/2021/1/1e/T--Wageningen_UR--wetlab_QS19.png)

**Figure 10.** Co-culture of the **E. coli** reporter-GFP strain and the **P. putida** AHL producing strain (left) or the WT (right) as negative control.
The positive control/WT strain was spread over the plate and drops of different dilutions of the reporter strain were added on top. Pictures of the green fluorescence were made.

[![](https://static.igem.org/mediawiki/2021/6/68/T--Wageningen_UR--Icon_proximity.png)](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/QuorumSensing)

###### Proximity-dependent kill switch

#### 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.

##### Amino acids exchange

Following a [preliminary study](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/Auxotrophy) two amino acids were selected for the establishment of the cross-feeding community: tryptophan (Trp) and tyrosine (Try). Specifically,
*P. putida* was chosen to be auxotrophic for Tyr and overproducing Trp, while *E. coli* was selected to be auxotrophic for Tyr and overproducing Trp.

Overall, the single mutant strains *P. putida* EM42 *ΔtyrA* (partial auxotroph for Tyr), *P. putida* EM42 *ΔphhAB* (partial auxotroph for Tyr), *E. coli ΔtrpD*
(auxotroph for Trp) and *E. coli ΔtyrR* (overproducer of Tyr) were obtained. Nevertheless, due to time constrains, the full cross-feeder strains could not be created. Anyway,
via a biosensor experiment, overproduction was studied in the single mutants *P. putida* EM42 *ΔtyrA* and *E. coli ΔtyrR*. *P. putida* EM42 *ΔtyrA* and
*E. coli ΔtyrR* were found to overproduce Trp and Tyr respectively. Overproduction of Trp and Tyr was detected to be in high enough quantities to support the growth of the *E. coli*
auxotrophic strains used as biosensors in the experiment (see figure 11). These preliminary results indicate that both *P. putida* EM42 *ΔtyrA* and *E. coli ΔtyrR* could sustain
the minimal growth of the cross-feeding partner.

![](https://static.igem.org/mediawiki/2021/9/9e/T--Wageningen_UR--Auxotrophy_graph_science.jpeg)

**Figure 11.** The overproduction of Tyr by *E. coli* and Trp by the *P. putida* was shown to sustain minimal growth of the auxotrophic biosensor *E. coli*
strains for the respective amino acid. Dashed lines indicate minimal concentration of the amino acid needed for growth.

Following a [preliminary study](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/Auxotrophy) two amino acids were selected for the establishment of the cross-feeding community: tryptophan (Trp) and tyrosine (Try). Specifically,
*P. putida* was chosen to be auxotrophic for Tyr and overproducing Trp, while *E. coli* was selected to be auxotrophic for Tyr and overproducing Trp.

Overall, the single mutant strains *P. putida* EM42 *ΔtyrA* (partial auxotroph for Tyr), *P. putida* EM42 *ΔphhAB* (partial auxotroph for Tyr), *E. coli ΔtrpD*
(auxotroph for Trp) and *E. coli ΔtyrR* (overproducer of Tyr) were obtained. Nevertheless, due to time constrains, the full cross-feeder strains could not be created. Anyway,
via a biosensor experiment, overproduction was studied in the single mutants *P. putida* EM42 *ΔtyrA* and *E. coli ΔtyrR*. *P. putida* EM42 *ΔtyrA* and
*E. coli ΔtyrR* were found to overproduce Trp and Tyr respectively. Overproduction of Trp and Tyr was detected to be in high enough quantities to support the growth of the *E. coli*
auxotrophic strains used as biosensors in the experiment (see figure 11). These preliminary results indicate that both *P. putida* EM42 *ΔtyrA* and *E. coli ΔtyrR* could sustain
the minimal growth of the cross-feeding partner.

![](https://static.igem.org/mediawiki/2021/9/9e/T--Wageningen_UR--Auxotrophy_graph_science.jpeg)

**Figure 11.** The overproduction of Tyr by *E. coli* and Trp by the *P. putida* was shown to sustain minimal growth of the auxotrophic biosensor *E. coli*
strains for the respective amino acid. Dashed lines indicate minimal concentration of the amino acid needed for growth

[![](https://static.igem.org/mediawiki/2021/9/9d/T--Wageningen_UR--Icon_auxotrophy.png)](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/Auxotrophy)

###### Auxotrophy

##### Carbon-source dependency between *E. coli* and *P. putida*

![](https://static.igem.org/mediawiki/2021/8/89/T--Wageningen_UR--wetlab_auxotrophy8.png)

**Figure 12.** Graphical representation of the ratios of *P. putida* and *E. coli* in co-culture at t0 and t24. Each dark circle indicates a single replicate,
while the bars indicate the mean of replicates at the time points indicated below the graph. The letter “P” indicates *P. putida*, while the letter “E” indicates *E. coli*.
t0 = 0 hours, t24 = 24 hours. Only significant differences are plotted and are indicated with a \*\* (\*\*p value < 0.01).

Carbon-source dependency was included to confer an additional layer of safety to the system. In this context, *P. putida* was engineered to rely solely on *E. coli* for the carbon-source
necessary to accumulate biomass. *E. coli* was shown to excrete acetate both during anaerobic and aerobic growth, and *P. putida* is known to grow on acetate as sole carbon-source.
Thus, a *P. putida* strain solely reliant on acetate for biomass generation was used in this project. This strain presents a double knock-out for the operon *gtsABCD* (encoding the
ATP-dependent ABC glucose transporter) and for the gene *gcd* (encoding the glucose dehydrogenase. The two knock-out are reported to impede the uptake of glucose by preventing its transport
through the inner membrane mediated by the ABC transporter and by blocking the peripheral oxidative route, which, through the action of Gcd, converts glucose to the intermediate D-gluconate.

Most importantly, co-culture experiments showed that *P. putida* EM42 significantly increased in abundance after 24 hours of co-culture with *E. coli* (Figure 12). Conversely, the co-culture of
*P. putida* EM42 *Δgts Δgcd* and *E. coli* was shown not to significantly differ from the initial inoculation ratio after 24 hours of growth (Figure 12). *P. putida* EM42
*Δgts Δgcd* was postulated to maintain a lower frequency in the population due to its dependence on the acetate secreted by *E. coli*.

Overall, carbon-source dependency was shown not only to serve as an ulterior layer of safety, but to successfully maintain the initial inoculation ratio of the two bacterial species after 24
hours of co-culture.

Carbon-source dependency was included to confer an additional layer of safety to the system. In this context, *P. putida* was engineered to rely solely on *E. coli* for the carbon-source
necessary to accumulate biomass. *E. coli* was shown to excrete acetate both during anaerobic and aerobic growth, and *P. putida* is known to grow on acetate as sole carbon-source.
Thus, a *P. putida* strain solely reliant on acetate for biomass generation was used in this project. This strain presents a double knock-out for the operon *gtsABCD* (encoding the
ATP-dependent ABC glucose transporter) and for the gene *gcd* (encoding the glucose dehydrogenase. The two knock-out are reported to impede the uptake of glucose by preventing its transport
through the inner membrane mediated by the ABC transporter and by blocking the peripheral oxidative route, which, through the action of Gcd, converts glucose to the intermediate D-gluconate.

Most importantly, co-culture experiments showed that *P. putida* EM42 significantly increased in abundance after 24 hours of co-culture with *E. coli* (Figure 12). Conversely, the co-culture
of *P. putida* EM42 *Δgts Δgcd* and *E. coli* was shown not to significantly differ from the initial inoculation ratio after 24 hours of growth (Figure 12). *P. putida* EM42
*Δgts Δgcd* was postulated to maintain a lower frequency in the population due to its dependence on the acetate secreted by *E. coli*.

Overall, carbon-source dependency was shown not only to serve as an ulterior layer of safety, but to successfully maintain the initial inoculation ratio of the two bacterial species after 24
hours of co-culture.

![](https://static.igem.org/mediawiki/2021/8/89/T--Wageningen_UR--wetlab_auxotrophy8.png)

**Figure 12.** Graphical representation of the ratios of *P. putida* and *E. coli* in co-culture at t0 and t24. Each dark circle indicates a single replicate,
while the bars indicate the mean of replicates at the time points indicated below the graph. The letter “P” indicates *P. putida*, while the letter “E” indicates *E. coli*.
t0 = 0 hours, t24 = 24 hours. Only significant differences are plotted and are indicated with a \*\* (\*\*p value < 0.01).

[![](https://static.igem.org/mediawiki/2021/9/9d/T--Wageningen_UR--Icon_auxotrophy.png)](https://2021.igem.org/Team%3AWageningen_UR/Wetlab/Auxotrophy)

###### Auxotrophy

* ###### References

  *arrow\_downward*

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  4. Z. Tan, Air Pollution and Greenhouse Gases. Springer, 2014.
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  6. J. M. Wehrfritz, A. Reilly, S. Spiro, and D. J. Richardson, “Purification of hydroxylamine oxidase from Thiosphaera pantotropha. Identification of electron acceptors that couple heterotrophic nitrification to aerobic denitrification,” FEBS Lett., vol. 335, no. 2, pp. 246–250, 1993.
  7. and K. M. L. Caranto, Jonathan D., “Nitric Oxide Is an Obligate Bacterial Nitrification Intermediate Produced by Hydroxylamine Oxidoreductase,” Proc. Natl. Acad. Sci. U. S. A., vol. 114, no. 31, pp. 8217–22, 2017.
  8. D. E. Liu, X., Shu, Z., Sun, D., Dang, Y., & Holmes, “Heterotrophic nitrifiers dominate reactors treating incineration leachate with high free ammonia concentrations,” ACS Sustain. Chem. Eng., vol. 6, no. 11, pp. 15040–15049, 2018.
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  11. M. Miyahara et al., “Potential of aerobic denitrification by pseudomonas stutzeri TR2 to reduce nitrous oxide emissions from wastewater treatment plants,” Appl. Environ. Microbiol., vol. 76, no. 14, pp. 4619–4625, Jul. 2010.
  12. K. Medhi, A. Singhal, D. Chauhan, I. T.-B. technology, and undefined 2017, “Investigating the nitrification and denitrification kinetics under aerobic and anaerobic conditions by Paracoccus denitrificans ISTOD1,” Elsevier.
  13. T. S, “Expression using the T7 RNA polymerase/promoter system,” Curr. Protoc. Mol. Biol., vol. Chapter 16, no. 1, Jul. 2001.
  14. Y. Pan, B.-J. Ni, P. L. Bond, L. Ye, and Z. Yuan, “Electron competition among nitrogen oxides reduction during methanol-utilizing denitrification in wastewater treatment,” 2013.
  15. S. Wenk et al., “An ‘energy‐auxotroph’ Escherichia coli provides an in vivo platform for assessing NADH regeneration systems,” Biotechnol. Bioeng., vol. 117, no. 11, pp. 3422–3434, Nov. 2020.
  16. R. K. Bennett et al., “Expression of soluble methane monooxygenase in Escherichia coli enables methane conversion,” bioRxiv, p. 2021.08.05.455234, Aug. 2021.
  17. H. J. Kim et al., “Biological conversion of methane to methanol through genetic reassembly of native catalytic domains,” Nat. Catal. 2019 24, vol. 2, no. 4, pp. 342–353, Apr. 2019.
  18. B. W. Hütsch, “Tillage and land use effects on methane oxidation rates and their vertical profiles in soil,” Biol. Fertil. Soils, vol. 27, no. 3, pp. 284–292, Jul. 1998.
  19. F. Y. H. Chen, H. W. Jung, C. Y. Tsuei, and J. C. Liao, “Converting Escherichia coli to a Synthetic Methylotroph Growing Solely on Methanol,” Cell, vol. 182, no. 4, pp. 933-946.e14, Aug. 2020.
  20. H. Yurimoto, N. Kato, and Y. Sakai, “Assimilation, dissimilation, and detoxification of formaldehyde, a central metabolic intermediate of methylotrophic metabolism,” Chem. Rec., vol. 5, no. 6, pp. 367–375, Jan. 2005.
  21. K. J. Denby et al., “The mechanism of a formaldehyde-sensing transcriptional regulator,” Sci. Rep., vol. 6, no. 1, pp. 1–15, Dec. 2016.
  22. B. M. Woolston, T. Roth, I. Kohale, D. R. Liu, and G. Stephanopoulos, “Development of a formaldehyde biosensor with application to synthetic methylotrophy,” Biotechnol. Bioeng., vol. 115, no. 1, pp. 206–215, Jan. 2018.
  23. K. Gerdes, L. K. Poulsen, A. K. Nielsen Novozymes, and J. Martinussen, “The hok killer gene family in gram-negative bacteria Regulation of stringent response in E.coli View project FAST and BM4SIT and the ESA-ITN project View project,” 1990.
  24. M. B. Miller and B. L. Bassler, “Quorum Sensing in Bacteria,” Annu. Rev. Microbiol., vol. 5, no. 1, pp. 165–199, 2001.
  25. W. R. J. D. Galloway, J. T. Hodgkinson, S. D. Bowden, M. Welch, and D. R. Spring, “Quorum sensing in Gram-negative bacteria: Small-molecule modulation of AHL and AI-2 quorum sensing pathways,” Chem. Rev., vol. 111, no. 1, pp. 28–67, 2011.
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##### About Cattlelyst

Cattlelyst is the name of the iGEM 2021 WUR team. Our name is a mix of 1) our loyal furry friends, cattle, and 2) catalyst, which is something that increases the rate of a reaction.
We are developing “the something” that converts the detrimental gaseous emissions of cattle, hence our name Cattlelyst.

Are you curious about our journey? We have written about our adventures in our blog, which you can find here:

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