iGEM 2021 — Team MSP-Maastricht

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

# Team:MSP-Maastricht/Notebook

![](https://static.igem.org/mediawiki/2021/b/ba/T--MSP-Maastricht--logoBrown.png)

* [Home](https://2021.igem.org/Team%3AMSP-Maastricht)

* Team
  + [Team](https://2021.igem.org/Team%3AMSP-Maastricht/Team)
  + [Attributions](https://2021.igem.org/Team%3AMSP-Maastricht/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AMSP-Maastricht/Collaborations)

* Project
  + [Contribution](https://2021.igem.org/Team%3AMSP-Maastricht/Contribution)
  + [Description](https://2021.igem.org/Team%3AMSP-Maastricht/Description)
  + [Engineering](https://2021.igem.org/Team%3AMSP-Maastricht/Engineering)
  + [Experiments](https://2021.igem.org/Team%3AMSP-Maastricht/Experiments)
  + [Notebook](https://2021.igem.org/Team%3AMSP-Maastricht/Notebook)
  + [Proof Of Concept](https://2021.igem.org/Team%3AMSP-Maastricht/Proof_Of_Concept)
  + [Proposed Implementation](https://2021.igem.org/Team%3AMSP-Maastricht/Implementation)
  + [Model](https://2021.igem.org/Team%3AMSP-Maastricht/Model)
  + [Results](https://2021.igem.org/Team%3AMSP-Maastricht/Results)

* [Parts](https://2021.igem.org/Team%3AMSP-Maastricht/Parts)

* [Safety](https://2021.igem.org/Team%3AMSP-Maastricht/Safety)

* [Human Practices](https://2021.igem.org/Team%3AMSP-Maastricht/Human_Practices)
  + [Human Practices](https://2021.igem.org/Team%3AMSP-Maastricht/Human_Practices)
  + [Collaborations](https://2021.igem.org/Team%3AMSP-Maastricht/Collaborations)

* [Education & Communication](https://2021.igem.org/Team%3AMSP-Maastricht/Communication)

* Awards
  + [Education](https://2021.igem.org/Team%3AMSP-Maastricht/Education)
  + [Entrepreneurship](https://2021.igem.org/Team%3AMSP-Maastricht/Entrepreneurship)
  + [Hardware](https://2021.igem.org/Team%3AMSP-Maastricht/Hardware)
  + [Inclusivity](https://2021.igem.org/Team%3AMSP-Maastricht/Inclusivity)
  + [Model](https://2021.igem.org/Team%3AMSP-Maastricht/Model)
* [Documentary](https://2021.igem.org/wiki/index.php?title=Team:MSP-Maastricht/Documentary)

# Notebook

### Week 1: Introductions and Idea Pitches

Once our team had been determined, we began with the first steps of our project. Our first meeting consisted of introducing ourselves, our courses and our background in science. This was then followed by pitching of ideas for our potential project

### Week 2: Team and Role Assignment

Now that we had gotten to know each other online, it was time to create teams and assign roles. We determined that seven subgroups were necessary to complete this project: Organization, External Communication, Programming, Scientific Communication, Journal, Funding, and the Drylab/Wetlab subgroups. Each team member chose 2-3 subgroups to be a part of. We also assigned a social media manager (Saskia) and team roles such as Team Managers (Anna and David), Team Leader (Tim/Kevin), and Secretary (Maaike).

### Week 3: Goals Were Determined

To decide what type of project we wanted to make, different aspects were considered and certain project ideas were ruled out as either impractical, unnecessary or already attempted in past iGEM projects. This week our social media introductions also took place, where we were introduced ourselves to those that followed our Instagram account.

### Week 4: Meeting Times and Topics Were Chosen

A presentation of topics occurred where 3 ideas were researched and weighed against each other by the dry lab team. From there they were presented to the rest of the team where we then voted to choose the topic of stopping ruminant methane emissions.

### Week 5: Began Collaborations and Pre-Requisites Were Distributed

The first teams were contacted by our external relations team and our first meeting was held (with Wageningen's graduate team). The iGEM deliverables were also divided up between the sub-teams in order to ensure that everything was covered.

### Week 6: iGEM Registration and Medal Criteria Distribution

With our team officially registered for iGEM, each subgroup started doing research on what projects they wanted to complete. This includes looking into the individual medal and award criteria and determining which ones we would attempt to fulfill.

### Week 7: Logo Design and First Monthly Report

The next big step in the identification of our team came about as our social media team created our logo. This was also when the first set of weekly reports were submitted and everyone got an overview of the whole project.

### Week 8: First In-Person Meeting for iGEM Welcome Ceremony

As iGEM got set up, so did we. Finally, with the lifting of some Covid restrictions, we were able to meet some of our team mates face to face in order to watch the iGEM welcoming ceremony. The Journal team met with writing expert Joop Westra for the first time who helped develop the workshops.

### Week 9: Exam Week

With most of the team busy preparing for exams, minimal work was done this week. Mostly consisting of report backs from collaboration meetings including the Uppsala, Tirupati and Pune teams.

### Week 10: Project Period Planning

A meeting was held to create a plan for the weeks to come during project period. This is a 4 week interval schedule in the MSP program where students focus on completing projects, then writing a report and doing a presentation about them. Monthly feedback was also given.

### Week 11: First In Person Meeting and Second Monthly Report

We had our first in person meeting and socializing event, where progress and ideas were discussed. The second monthly reports were also handed in to the organization team. Lab work also has begun:

15.06.21
Transformation with LB broth without Kanamycin. 100ul of culture were used on the plates.
16.06.21
Agar plates creation and plating. Competent cell creation.
17.06.21
Remaking of transformed cells

### Week 12: Design Detailing

In person meetings allowed for a better flow of creative ideas and designs started to arise. Finally our project name was chosen to be MethaGone. In hopes of showing off our style at the Jamboree we also started designing our apparel and masks.

21.06.21
From the transformed cells, two colonies were chosen each for overnight cultures in LB + Kanamycin. Total of 8 vials - for miniprep

22.06.21
Band present in the gel. Overnight cultures were generated from the corresponding colony for PCR. Primers resuspension and working stock creation based specification sheets by IDT.

24.06.21
Resuspension of the gBlock constructs based on specification sheets given by IDT.

### Week 13: New Calendar and More Lab Work

Moving to Taskade had a positive impact on our team and enabled us to be more organized in assigning tasks. The lab team continued working to linearize the plasmid (and we made some creative agar plates on the side). The programming and journal team created a website in order to help the communication with the journal participants. The construct designs were completed and the genes present in the lab.

### Week 14: Waganingen Meetup and Project Period Report

An online event was organized by Wageninger and included all the teams from around the Netherlands. All teams gave a project pitch and then listened to guest speakers. With multiple members missing because of this and other circumstances, no meeting was held that week. The Journal team also wrote and compiled a report of our project up until this point as required by the university.

8.7.21
Successful Transformation of the genes, colony selection and overnight culture.

9.7.21
Duplex gene combos via Gibson Assembly done, transformation and plating carried out.

10.07.21
Single gene transformations were successful on plates however DNA wasn’t clearly present on the gel. Duplex gene transformations (Gene + Mbb2) had successful growth, 1 plate did not.

### Week 15: G-Block Arrive and Summer Planning

Finally, the long awaited G-Blocks arrived from IDT and the lab team was ready to start modifying. Within the week they make competent cells of the bacteria, suspended and inserted DNA as well as inserted multiple genes into the same bacteria, both those that coded for bromoform and peroxide production. Monthly reports were due again and progress was shared between the subgroups, the goals for each subgroup over the summer (month of July) were also established during this meeting.

11.07.21
Miniprep of Mbb4 and combinations of mbb2+mbb1/mbb4/ccMbb3. Restriction digestion and gel electrophoresis

12.07.21
More agar plates containing Kanaymcin. Linearization of pET39b+ ( further stock). Neb Hifi Assembly:

Mbb2+Mbb1+Mbb4
Mbb2+Mbb1+ccMbb1
Mbb2+Mbb1+ccMbb3
Mbb2+Mbb4+ccMbb1
Mbb2+Mbb4+ccMbb3

Competent cell transformation and plating on agar.

13.07.21
Minipreps and Restriction Digest using XbaI and NdeI. Visualization on Gel.

14.07.21
New working primer stock solution for other primers. Lb broth and agar plates creation.

### Week 16: Brightlands Funding Pitch and the OJS

The journal website was officially up and running with a fully functioning Open Journaling System (OJS). This was an exciting step for the MSP Vector as it allowed a platform to organize submissions and distribution of articles for the journal. From the Science Communication team, filming, editing and feedback was underway for the promotional video that was due in the upcoming week. A pitch was also done to the Brightlands Venture Capital which was a great experience for the funding team and earned us €250.

### Week 17: Moving

This week was intensely disruptive as our university labs were moving from the Brightlands campus to a Maastricht Campus. This caused our lab to be split and ment that we had to organize to keep all the supplies we needed from being moved to the new labs. Our project remained at the same campus due to regulation restrictions and for the sake of consistency.

### Week 18: Promotional Video

The promotional video was worked on and finalized this week thanks to the hard work of the science communication team. Filming at the lab and at a cattle farm in the area produced some amazing shots that made this possible. This video can be seen at the bottom of the home page.

### Week 19: Managerial Involvement and Safety meeting

Due to holidays, members from the organizational team became more directly involved in the subgroups in order to get an overview of their progress and upcoming goals. A meeting with RIVM which is the dutch National Institute for Public Health and the Environment (Rijksinstituut voor Volksgezondheid en Milieu) was organized to talk about GMO safety.
09.08.21
Lb broth creation, overnight culture for competent cells. A HiFi assembly was created.

11.08.21
Agar plates. Gene transformations and plating.

12.8.21
No colonies grew overnight. A new hifi assembly was conducted with a different linearized plasmid.

13.8.21
Single colonies were re-plated. The genes were PCR with specific gene primers.

### Week 20: GMO and Farmer Survey Launched

Simultaneously, a survey directed at farmers in collaboration with iGEM Uppsala, and a survey directed at the general public about GMOs in collaboration with iGEM Tirupati, Pune and Aachen was launched. The workshops for the journal were uploaded to youtube.

16.08.21
Purification and miniprep of genes and gel. Followed by gel extraction. Gel was not showing good results. PCR of the genes.

17.08.21
Ran a gel from PCR, and got better results. Gel extraction of mbb2, not strong concentration.

18.08.21
Gel extractions concentrations were not high enough. Another gel with the previous PCR was done and gel extractions were conducted. Liquid cultures were made with single cultures.

20.08.21
PCR of Mbb1 & Mbb4 were redone since clear bands were not present. Modification of the PCR annealing temperature. Miniprepp of other single gene constructs.

### Week 21: Started Business Plan

A subset of the funding team began to create a business plan in order to fulfil the criteria for the Entrepreneurship medal. This involved brainstorming to create schematics in canva that can be seen on the page. These schematics were then written up into the pdf business plan.

23.08.21
Restriction digestion of the genes, single genes in pET39b and followed by a gel electrophoresis.

24.08.21
Gel electrophoresis was carried out and the single PCRed genes were checked following PCR clean up.

26.08.21
Massive gel, with Restriction Digestion genes, PCRed genes and the original genes to compare.

27.08.21
ccMbb1 and ccMbb3 appeared to be correct in the gel, sent off for sequencing

### Week 22: Crowdfunding Went Online

After getting input from some advisors within the crowdfunding field, it was time to launch our crowdfunding campaign. The original soft launch was to persuade our warm contact (people we are personally connected to) to donate and later it was more widely distributed to the general public.

1.09.21
Re-did the single genes constructs. They were transformed and plated on kanamycin agar plates.

2.09.21
Growth was not detected on the plates therefore they were left to go further.

### Week 23: Swiss School Workshop

This workshop was run by two of our members over Zoom to a class of teenagers in Switzerland. This workshop created an environment to debate about GMOs and other ethical issues while also teaching a higher level of English to the German-speaking students.

6.09.21
Colonies were present on the plates and an overnight culture was created.

7.09.21
The overnight culture of the single genes was miniprepped and the pieces were sent for sequencing

### Week 24: Card Game and Peer Review for Journal

The card game for our Science Communication was completed and a test round was then played by our team members. It was then left in the facility common room for other people to play and give feedback on. The journal peer-review pairs were also assigned and sent out this week. Each team was given 2 other teams to peer review and then received 2 back that could then be used to make improvements on their article before submission.

12.09.21
We redid the triplex gene PCR and a Gibson assembly and transformation into DH5a competent cells. They were then plated on Kanamycin plates and left to grow in the incubator.

15.09.21
The plates were left to grow for 3 days to have a great amount of colonies for testing. From this we extracted the cells and created an overnight culture.

16.09.21
We miniprepped the overnight culture and then we ran a restriction digestion to visualize the bands. Due to excess noise we were going to repeat it the following lab day.

18.09.21
We redid the gel however the bands were not in the proper location, which meant two things: either our restriction digest was not successful or our genes do not contain the triplex genes. We selected 3 colonies of each gene combination and grew them in an overnight culture.

### Week 25: Invited to Complete Blog Posts and Crowdfunding Video Was Made

Biotechnologie invited our team to create a series of 6 blog posts documenting our experience in iGEM as well as explaining our project. We completed and uploaded our promotional video with a funding task to the crowdfunding platform as we opened it up to the wider public.

27.09.21
We received the triplex gene sequences back and they contained the sequence of the original vector. Therefore we opted to switch with duplex genes only, they would all contain Mbb2 and the other respective gene.

28.09.21
We ran a PCR on our genes so that we could create the duplex combinations. Following this PCR we ran a Gibson Assembly and a transformation and plated the cells.

30.9.21
We were able to see growth on the plates and we created an overnight culture from 3 different colonies.

01.10.21
We mini-prepped the colonies and we sent them off to sequencing to gather the data and check if our duplex combination transformations were correct.

04.10.21
We continued with our single gene constructs; Mbb1 and ccMbb1, we replated these from the successful colonies so that we could generate more. We left these growing overnight on plates in the incubator.

### Week 26: Surveys Closed and Analyzed

The survey collaborations were close with a total of 275 responses for the GMO and 10 responses for the Farmers survey. The data was then taken by each team and analyzed separately. For the GMO survey, empirical data were generated and analyzed in graphs. For the farmer survey, each response was individually processed to address individual concerns.
Editing of the documentary has officially started requiring footage licenses to be requested. The final questions for the interviews with various farmers were finished.
A cost structure for future field testing was established in the financing team. Stakeholders were contacted that were mentioned in the business plan. A radio interview was pre-recorded.

07.10.21
We then selected a few and grew them in an overnight culture.

09.10.21
We miniprepped them and we prepared the solutions and buffers required to create an SDS-PAGE.

11.10.21
From here we treated our samples and placed them in a gel and ran it for 40 mins. Following this the gel was removed and we placed it in a staining solution overnight.

### Week 27: Video Presentation Preperation

The first blogpost for Biotechnologie was posted, which introduces our team and our project. Peer-reviewing was assigned within the group for different parts of our wiki texts. This week and A person was assigned to create a team that is in charge of a storyboard for the presentation video. With the final weeks of the project ddawing near, the journal team struggled to receive the final articles from the participants.

12.10.21
We then checked if any bands were present on the gel, however, the staining solution was too deep to visualize anything. We placed the gel in a destaining solution overnight.

13.10.21
Our duplex gene sequence results returned and unfortunately they did not represent the actual genes, only our original vector.

### Week 28: Disabled Children's workshop and Politician interview took place

Two interviews with politicians took place, one in person in the Hague, and one online in order to acquire footage and information for the documentary. A timeline was simultaneously created for the filming and editing of the presentation video. With all the equipment in place, the ruminal simulation was able to take place. A few of our members traced to a nearby slaughterhouse to acquire a cow's ruminal fluid and solids, and then brought that to the lab for testing in the rumen simulation.

### Week 29: Wiki freeze

This week marks the final stages of the iGEM competition with the wiki freeze approaching on the 21st of October. There is a rush to finalize the wiki pages as well as make the final edits on the documentary and the journal.

### Week 30: Attending the BeNaLux Mini Jamboree

We will be attending the mini jamboree hosted in Eindhoven. We plan to travel by train to the Eindhoven to meet other iGEM teams from the Netherlands and present our project.

### Week 31: iGEM judging will take place on the 5/11/2021

Our project is scheduled for judging on the 5th of November

![](https://static.igem.org/mediawiki/2021/9/99/T--MSP-Maastricht--Footer-full-logo.png)

## Sponsors

![Maastricht University logo](https://static.igem.org/mediawiki/2021/c/cf/T--MSP-Maastricht--Footer-UM-logo.png)

![Integrated DNA Technologies logo](https://static.igem.org/mediawiki/2021/8/88/T--MSP-Maastricht--Footer-IDT-logo.png)

![SnapGene logo](https://static.igem.org/mediawiki/2021/a/a7/T--MSP-Maastricht--Footer-SnapGene-logo.png)

![Brightlands Venture Partners logo](https://static.igem.org/mediawiki/2021/e/e7/T--MSP-Maastricht--Footer-Brightlands-logo.png)

![GenScript logo](https://static.igem.org/mediawiki/2021/f/fc/T--MSP-Maastricht--Footer-GenScript-logo.png)

![HadeCo logo](https://static.igem.org/mediawiki/2021/f/fc/T--MSP-Maastricht--Footer-imgB-logo-hadeco2.jpeg)

Instagram: [msp\_igem](https://www.instagram.com/msp_igem/)
Linked-in: [iGEM MSP-Maastricht](https://www.linkedin.com/company/msp-igem-maastricht/)
Youtube: [Geneducation](https://www.youtube.com/channel/UCpZQvRTiQNvqz9XcqDGK9pA/featured)
Facebook: [MSP iGEM](https://www.facebook.com/MSPIGEM)
Tik-tok: msp\_igem

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

# Team:MSP-Maastricht/Engineering

![](https://static.igem.org/mediawiki/2021/b/ba/T--MSP-Maastricht--logoBrown.png)

* [Home](https://2021.igem.org/Team%3AMSP-Maastricht)

* Team
  + [Team](https://2021.igem.org/Team%3AMSP-Maastricht/Team)
  + [Attributions](https://2021.igem.org/Team%3AMSP-Maastricht/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AMSP-Maastricht/Collaborations)

* Project
  + [Contribution](https://2021.igem.org/Team%3AMSP-Maastricht/Contribution)
  + [Description](https://2021.igem.org/Team%3AMSP-Maastricht/Description)
  + [Engineering](https://2021.igem.org/Team%3AMSP-Maastricht/Engineering)
  + [Experiments](https://2021.igem.org/Team%3AMSP-Maastricht/Experiments)
  + [Notebook](https://2021.igem.org/Team%3AMSP-Maastricht/Notebook)
  + [Proof Of Concept](https://2021.igem.org/Team%3AMSP-Maastricht/Proof_Of_Concept)
  + [Proposed Implementation](https://2021.igem.org/Team%3AMSP-Maastricht/Implementation)
  + [Model](https://2021.igem.org/Team%3AMSP-Maastricht/Model)
  + [Results](https://2021.igem.org/Team%3AMSP-Maastricht/Results)

* [Parts](https://2021.igem.org/Team%3AMSP-Maastricht/Parts)

* [Safety](https://2021.igem.org/Team%3AMSP-Maastricht/Safety)

* [Human Practices](https://2021.igem.org/Team%3AMSP-Maastricht/Human_Practices)
  + [Human Practices](https://2021.igem.org/Team%3AMSP-Maastricht/Human_Practices)
  + [Collaborations](https://2021.igem.org/Team%3AMSP-Maastricht/Collaborations)

* [Education & Communication](https://2021.igem.org/Team%3AMSP-Maastricht/Communication)

* Awards
  + [Education](https://2021.igem.org/Team%3AMSP-Maastricht/Education)
  + [Entrepreneurship](https://2021.igem.org/Team%3AMSP-Maastricht/Entrepreneurship)
  + [Hardware](https://2021.igem.org/Team%3AMSP-Maastricht/Hardware)
  + [Inclusivity](https://2021.igem.org/Team%3AMSP-Maastricht/Inclusivity)
  + [Model](https://2021.igem.org/Team%3AMSP-Maastricht/Model)
* [Documentary](https://2021.igem.org/wiki/index.php?title=Team:MSP-Maastricht/Documentary)

# Engineering Success

The process of implementing our ideas and concepts on making MethaGone a reality can be easily systematized in the framework of the DBTL cycle iterations.

![dbtl cycle](https://static.igem.org/mediawiki/2021/b/b1/T--MSP-Maastricht--dbtl_cycle.png)

## Spacer Complex

For our project, we aimed at inserting multiple haloperoxidases from two organisms, Asparagopsis
taxiformis, and Chondrus crispus. Initially, we wanted to insert three genes of A. taxiformis and
two of C. crispus. However, we realized that the genes themselves would be large and inefficient in
the Gibson assembly we wanted to carry out.

We wanted to be able to produce the proteins simultaneously so that we could increase our efficacy. By doing so our Mbb2 protein, which is a reactive
oxygen species generator, will be produced alongside the bromoform producing genes. Since all of them required ROS to be able to carry out their process. Adding spacers between the genes would give us the flexibility to create different combinations with these different genes.

As a solution, the Spacer system was developed — a simple sequence of nucleotides, which would be present
before and after each gene. In the end, it allows us to generate multiple gene combinations enabling us to test different types and create a graph of the
most effective genes. This would also lead to a reduction in the number of primers that are needed. Only
two sets of primers were required to PCR and prepared the genes for the Gibson Assembly as they had the
same spacers.

A RBS site was
engineered to allow us to simultaneously translate the protein in our different combinations. We also
implemented a restriction site XbaI in the Forward Spacer and NdeI in the back-spacer. By doing so, we
were able to use one set of restriction enzymes and able to separate the genes from the construct and detect
them in a gel in one reaction.

We then used the spacer system approach and our corresponding primers worked as desired and it gave the correct assembly, which was confirmed by gel electrophoresis and sequencing.

From this we confirmed that the Spacer System would be able to create different combinations of genes as well as different orders when using specific primers for the order that is desired.

## Gut in Glass

It was important for us to make "MethaGone" as safe as it can be. For that reason, the testing stage had to be very reminiscent of in vivo, in cow conditions. However, it is impossible not to take into account the unethical nature of this approach.

The solution was our development and simulation of the gastric tract of a cow - our Gut in Glass. This is an easy-to-build and, therefore, highly adaptive adjustment. A video guided tutorial was made to explain the rumen simulation set up, you can find it using the following [link](https://2021.igem.org/Team%3AMSP-Maastricht/Hardware).

Once set up, the rumen simulation provided a stable anaerobic environment, a suitable habitat for archaea and other microorganisms present in intestinal systems.

 Repeated testing over long time periods were needed to ensure that anaerobic conditions can be maintained for long experiment that need to be conducted overnight.

Leaks were found and fixed, and correct conduct was tested for working with the simulation, while disturbing the anaerobic conditions as minimal as possible.

Further additions of the simulation included adaptations for temperature measurement and a volume measurement technique.

In our experiment, we were successful at showing that there are differences in gas productions between the control simulation and the one where bromoform was added. With this information, we have confirmation that we are able to continue our project with this setup, testing more results and expanding the time-scale of the experiment. More information can be found at [Project Modeling "Gut in Glass" section](https://2021.igem.org/Team%3AMSP-Maastricht/Model).

![oxygenmeter](https://static.igem.org/mediawiki/2021/thumb/0/02/T--MSP-Maastricht--oxygenmeter.png/610px-T--MSP-Maastricht--oxygenmeter.png)

Oxygen Gas Meter confirming anaerobic conditions within the rumen simulation.

![](https://static.igem.org/mediawiki/2021/9/99/T--MSP-Maastricht--Footer-full-logo.png)

## Sponsors

![Maastricht University logo](https://static.igem.org/mediawiki/2021/c/cf/T--MSP-Maastricht--Footer-UM-logo.png)

![Integrated DNA Technologies logo](https://static.igem.org/mediawiki/2021/8/88/T--MSP-Maastricht--Footer-IDT-logo.png)

![SnapGene logo](https://static.igem.org/mediawiki/2021/a/a7/T--MSP-Maastricht--Footer-SnapGene-logo.png)

![Brightlands Venture Partners logo](https://static.igem.org/mediawiki/2021/e/e7/T--MSP-Maastricht--Footer-Brightlands-logo.png)

![GenScript logo](https://static.igem.org/mediawiki/2021/f/fc/T--MSP-Maastricht--Footer-GenScript-logo.png)

![HadeCo logo](https://static.igem.org/mediawiki/2021/f/fc/T--MSP-Maastricht--Footer-imgB-logo-hadeco2.jpeg)

Instagram: [msp\_igem](https://www.instagram.com/msp_igem/)
Linked-in: [iGEM MSP-Maastricht](https://www.linkedin.com/company/msp-igem-maastricht/)
Youtube: [Geneducation](https://www.youtube.com/channel/UCpZQvRTiQNvqz9XcqDGK9pA/featured)
Facebook: [MSP iGEM](https://www.facebook.com/MSPIGEM)
Tik-tok: msp\_igem

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

# Team:MSP-Maastricht/Experiments

![](https://static.igem.org/mediawiki/2021/b/ba/T--MSP-Maastricht--logoBrown.png)

* [Home](https://2021.igem.org/Team%3AMSP-Maastricht)

* Team
  + [Team](https://2021.igem.org/Team%3AMSP-Maastricht/Team)
  + [Attributions](https://2021.igem.org/Team%3AMSP-Maastricht/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AMSP-Maastricht/Collaborations)

* Project
  + [Contribution](https://2021.igem.org/Team%3AMSP-Maastricht/Contribution)
  + [Description](https://2021.igem.org/Team%3AMSP-Maastricht/Description)
  + [Engineering](https://2021.igem.org/Team%3AMSP-Maastricht/Engineering)
  + [Experiments](https://2021.igem.org/Team%3AMSP-Maastricht/Experiments)
  + [Notebook](https://2021.igem.org/Team%3AMSP-Maastricht/Notebook)
  + [Proof Of Concept](https://2021.igem.org/Team%3AMSP-Maastricht/Proof_Of_Concept)
  + [Proposed Implementation](https://2021.igem.org/Team%3AMSP-Maastricht/Implementation)
  + [Model](https://2021.igem.org/Team%3AMSP-Maastricht/Model)
  + [Results](https://2021.igem.org/Team%3AMSP-Maastricht/Results)

* [Parts](https://2021.igem.org/Team%3AMSP-Maastricht/Parts)

* [Safety](https://2021.igem.org/Team%3AMSP-Maastricht/Safety)

* [Human Practices](https://2021.igem.org/Team%3AMSP-Maastricht/Human_Practices)
  + [Human Practices](https://2021.igem.org/Team%3AMSP-Maastricht/Human_Practices)
  + [Collaborations](https://2021.igem.org/Team%3AMSP-Maastricht/Collaborations)

* [Education & Communication](https://2021.igem.org/Team%3AMSP-Maastricht/Communication)

* Awards
  + [Education](https://2021.igem.org/Team%3AMSP-Maastricht/Education)
  + [Entrepreneurship](https://2021.igem.org/Team%3AMSP-Maastricht/Entrepreneurship)
  + [Hardware](https://2021.igem.org/Team%3AMSP-Maastricht/Hardware)
  + [Inclusivity](https://2021.igem.org/Team%3AMSP-Maastricht/Inclusivity)
  + [Model](https://2021.igem.org/Team%3AMSP-Maastricht/Model)
* [Documentary](https://2021.igem.org/wiki/index.php?title=Team:MSP-Maastricht/Documentary)

# Experiments

## Brainstorming Stage

An article by Thapa et al. (2020), which found the genetic mechanism for bromoform production in various seaweed species inspired us. The suggestion that feeding cows seaweed, such as A. taxiformis would reduce the amount of methane (CH4) produced from cows (Roque et al., 2019) initiated our research and led us to Thapa et al. (2020). This was fascinating and we investigated further. The algae A. taxiformis contains specialized gland cells from which the bromoform is secreted as a defense mechanism against parasites and herbivores (Machando et al., 2016). The bromoform can be produced through a photosynthetic-derived pathway or via a fatty acid biosynthesis-derived pathway (Zhu et al.,2021). Thapa et al. (2020) and Thapa & Agarwal (2021) explored the usage of pentane-2,4-dione and heptane-2,4,6-trione as substrates for the synthesis of bromoform. The genes in question were mbb1 and mbb4 which had the highest levels of bromoform production. The mbb1 and mbb4 are vanadium-dependant haloperoxidases (VHPOs) and they require a reactive oxygen species as a cofactor to generate the bromoform. Thus the solution for the creation of the ROS was also present as a gene in A. taxiformis – mbb2. Furthermore, VHPOs were present in another algae species, namely Chondrus crispus (Thapa et al. 2020). Two bromoform producing genes were identified – CcVHPO1 and CcVHPO3 which were highly efficient. Therefore, once we had conceptualized and actualized our idea with the genes, we wanted to test forward with the designing process.

![Schematic of bromoform synthesis pathway](https://static.igem.org/mediawiki/2021/1/16/T--MSP-Maastricht--Project-Experiments-img1-bromoformPathway.png)

Hypothesized Pathway of Bromoform Biosynthesis with Substrates and Products in Green and Enzymes
shown in Yellow. Adapted from Zhu et al. (2021) and Thapa et al. (2020).

## Design Stage

The DNA sequence of the genes was retrieved from Genbank. From this we wanted to maximize the number of combinations with the minimal number of genes required to be processed. We wanted a device that would allow us to orient the genes in whichever pattern we wanted. To do so we create the Spacer concept, essentially a simple DNA sequence that does not code for any proteins, nonsense DNA, however that would contain valuable recognition sequences. It contained a ribosome binding site to maximize the efficiency of large, transcribed proteins. Along with the RBS there was a restriction site XbaI introduced for both gel electrophoresis detection and analysis as well as being able to insert the gene of interest after the spacer for usage if so required. Then we wanted another spacer to flank the end of the target gene of interest. The back spacer contained nonsense DNA that would not be able to produce a protein. Again, for testing purposes as well as inserting the genes, a restriction site was present, NdeI.

## Experimental Stage

For this we used several protocols. We followed this process:

* Creation of the competent cells – DH5a for cloning and BL21 for protein expression
* Assembly of the single genes in the DH5a cells. To maximize the usage of DNA that was generated we inserted the gene of interest into DH5a so that with a correct transformation using Gibson Assembly we would be able to generate plates containing that construct which we can use whenever and in an endless supply
* After every Gibson Assembly we always made sure that we would run an agarose gel to be able to analyze what we were able to construct.
* Following this we created our pattern system using the spacer technology to create our gene orientations
* Protein Secretion
* Protein Purification
* Testing in cow ruminal fluid (see [Model section](https://2021.igem.org/Team%3AMSP-Maastricht/Model))

## Chemically Competent Cells

**Materials**

* Overnight Culture of cells of your choice
* Ice-Cold 0.1M CaCl2
* Ice-Cold 0.1M CaCl2 +15% glycerol
* 50ml Falcon tubes
* Pre-chilled Eppendorf tubes

**Procedure**

1. Create an overnight culture in LB liquid without any antibiotics
2. Grow the cells in a incubating shaker (250 to 270rpm) at 37C until the OD600 of the cells is between
   0.4-0.6
3. Chill the cells on ice for 10 to 15 mins (maximum is 1 hour do not exceed this time) to prevent further
   growth
4. Spin down the cells in a 50ml Falcon tube at 4000 rpm for 10min at 4C
5. Remove the supernatant; from now work is to be carried on ice
6. Suspend the pellet with cold 10ml 0.1M CaCl2
7. Spin it again at 4000rpm at 4C for 10 min
8. Gently resuspend cells with cold 2ml 0.1M CaCl2 and 2ml of 15% glycerol
9. Allocate the solution (200ul) into different pre chilled Eppendorf tubes

## DPNI Treatment

**Materials**

* CR reactions
* DPNI vial

**Procedure**

1. Add 1µL of DpnI to finished 50µL PCR reactions (or .5µL to 25µL reactions). Pipet or invert to mix.
2. Incubate the mixture at 37°C for 1-2 hrs (depending on your paranoia or need to remove template DNA).
3. Alternatively, the solution can be left overnight at room temperature. Periodic mixing may aid digestion (but
   is unnecessary).
4. PCR cleanup or gel-purify the reaction for downstream processes. It's that easy!

## LB Medium

**Materials**

* Tryptone
* Yeast Extract
* NaCl
* Agar

**Procedure**

1. LB Agar Plates
2. Add the following to a 1000ml container:
3. 10 g tryptone (Fisher, cat. no. DF0123-17-3)
4. 5 g yeast extract (Fisher, cat. no. DF0127-17-9)
5. 5 g NaCl (Fisher, cat. no. S271-500)
6. 15 g agar (Fisher, cat. no. DF0140-07-4)
7. Adjust volume to 1000 ml with double-distilled water
8. Adjust to pH 7.0
9. Autoclave, let cool to about 60°C (cool enough to handle without gloves), and then pour into sterile plastic
   petri dishes (Fisherbrand, cat. no. 08-757-12)
10. Allow the plates to cool and solidify at room temperature
11. Store the plates inverted in a plastic sleeve up to 6 months at 4°C

**LB Broth**

1. Add the following to a 1000ml container:
2. 10 g tryptone (Fisher, cat. no. DF0123-17-3)
3. 5 g yeast extract (Fisher, cat. no. DF0127-17-9)
4. 5 g NaCl (Fisher, cat. no. S271-500)
5. Adjust volume to 1000 ml with double-distilled water
   Autoclave
6. Store indefinitely at room temperature

## Gel Extraction Kit

**Procedure - Extracted from QIAquick Gel Extraction Kit**

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg gel ~100 μl). The
   maximum amount of gel per spin column is 400 mg. For >2% agarose gels, add 6 volumes Buffer QG.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3
   min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the
   mixture is yellow (like Buffer QG without dissolved agarose). If the color of the mixture is orange or
   violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.
4. Add 1 gel volume isopropanol to the sample and mix.
5. Place a QIAquick spin column in a provided 2 ml collection tube or into a vacuum manifold. To bind DNA,
   apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum to the manifold until all
   the samples have passed through the column. Discard flow-through and place the QIAquick column back into
   the same tube. For sample volumes >800 μl, load and spin/apply vacuum again.
6. If DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 500 μl
   Buffer QG to the QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place
   the QIAquick column back into the same tube.
7. To wash, add 750 μl Buffer PE to QIAquick column and centrifuge for 1 min or apply vacuum. Discard
   flow-through and place the QIAquick column back into the same tube. Note: If the DNA will be used for
   salt-sensitive applications (e.g., sequencing, bluntended ligation), let the column stand 2–5 min after
   addition of Buffer PE. Centrifuge the QIAquick column in the provided 2 ml collection tube for 1 min to
   remove residual wash buffer.
8. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
9. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAquick
   membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 μl Buffer EB to the
   center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the
   addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase
   the yield of purified DNA.
10. If purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA.
    Mix the solution by pipetting up and down before loading the gel.

## General Transformation

**Materials**

* Competent E.coli Cells - homemade or commercial
* DNA for insertion

**Procedure**

- Thaw a tube of Competent E.coli cells on ice for 10 mins
- Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix
  cells and DNA. Do not vortex.
- Place the mixture on ice for 30 minutes. Do not mix.
- Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
- Place on ice for 5 minutes. Do not mix.
- Pipette 950 µl of room temperature SOC or (LB could work in this case) into the mixture.
- Place at 37°C for 60 minutes in the shaker-incubator.
- Warm selection plates to 37°C - the ones that contain Kanamycin
- Spread 50-100 µl from the Eppendorf container onto a selection plate (make multiple) and incubate overnight at
  37°C.

## Gibson Assembly

**Materials**

NEBuilder HiFi DNA Assembly
DNA fragments

**Procedure**

1. Mix 10ng – 100ng of each of the DNA fragments together into 5ul total volume. The length of each
   fragment
   and the concentration of the fragments must be taken into account
2. If using the 2x Gibson Mater Mix from NEB – add 10ul of total DNA to a 10ul Gibson Assembly Master
   Mix
3. Mix well by pipetting
4. Incubate the reaction at 50C for 1 hour

## Glycerol Stock

**Materials**

* 50% glycerol solution
* Desired cell culture to create stock

**Procedure**

1. Overnight liquid culture creation
2. After you have bacterial growth, add 500ul of the overnight culture to 500ul of 50% glycerol in a 2ml
   screw
   top tube
3. Dilute pure glycerol in distilled water to create a 50% glycerol solution - so 50:50 (
   glycerol:water)
4. Freeze the glycerol stock at -80C

## Gel Electrophoresis

**Materials**

* Standard 1% Agarose Gel
* Agarose: 1g
* TAE: 100ml
* GelRed® Nucleic Acid Gel Stain: 2-3ul

**Procedure**

Pouring a Standard 1% Agarose Gel:

Measure 1g of agarose1. Mix agarose powder with 100ml 1xTAE in a conical flask
2. Microwave for 1-3 min until the agarose is completely dissolved – don’t let it overboil however make
   sure
3. that there are no white particles in it; caution the contain is hot use thermal gloves
4. Let the agarose solution cool down to 50C – occasionally test it with the back of your hand for 5 secs
5. Add 2-3ul GelRed Stain to the agarose solution
6. Pour the agarose into a gel tray with a well comb
7. Let the agarose gel to sit at room temperature for 20-30mins until the gel is solid

Sample Loading:

1. Add (loading dye – purple or orange) around 2ul of the dye should suffice
2. Once the gel has fully solidified place it in the box.
3. Fill gel with 1xTAE until gel is submerged till the mark on the box.
4. Load the Gene Ladder in the first lane of the gel
5. It is recommended but not required to take a space lane where it is not filled in
6. Run the gel at 80-150V until the process is approximately 75%-80% of the way down the gel, keep it running
   for 1-1.5hours
7. Turn OFF the power, disconnect the electrodes and remove gel
8. Using (MACHINE) with UV light device you are able to visualize the DNA fragments

## Miniprep Extraction

**Procedure - Retrieved from Qiagen Miniprep Kit**

1. Pellet by centrifuging for 3min at 3000rpm/ 6800g’s. Resuspend pellet bacterial cells in 250 μl Buffer
   P1
   and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. No cell clumps
   should be visible after resuspension of the pellet.
2. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Mix gently by inverting the
   tube. Do
   not vortex, because this will result in shearing of genomic DNA and contamination of plasmid. Continue
   inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction
   to
   proceed for more than 5 min.
3. Add 350 μl Buffer N3. Mix immediately and thoroughly by inverting the tube 4–6 times. To avoid localized
   precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes
   (e.g., ≥5 ml) may require inverting up to 10 times. The solution should become cloudy.
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet
   will
   form.
5. Apply 800 μl of the supernatant from step 4 to the QIAprep 2.0 Spin Column by pipetting.
6. Centrifuge for 30–60 s. Discard the flow through.
7. Recommended: Wash the QIAprep 2.0 Spin Column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s.
   Discard the
   flow through. This step is necessary to remove trace nuclease activity when using endA+ strains, such as
   the JM
   series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or
   high
   carbohydrate content. Host strains, such as XL-1 Blue and DH5α, do not require this additional wash step.
8. Wash QIAprep 2.0 Spin Column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
9. Discard the flow through, and centrifuge at full speed for an additional 1 min to remove residual wash
   buffer.
   Important: Residual wash buffer will not be completely removed unless the flow through is discarded before
   this
   additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
10. Place the QIAprep 2.0 Spin Column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer
    EB (10 mM
    Tris·Cl, pH 8.5) or water to the center of each QIAprep 2.0 Spin Column, let stand for 1 min, and
    centrifuge for 1
    min.

## Polymerase Chain Reaction

**Procedure**

![](https://static.igem.org/mediawiki/2021/thumb/3/3d/T--MSP-Maastricht--experiment-table1.png/800px-T--MSP-Maastricht--experiment-table1.png.jpeg)

![](https://static.igem.org/mediawiki/2021/thumb/0/07/T--MSP-Maastricht--experiment-table2.png/800px-T--MSP-Maastricht--experiment-table2.png.jpeg)

## Restriction Digestion

Procedure
1 unit of restriction enzyme will completely digest 1ug of substrate DNA in a 50ul reaction in 60 minutes.

 ![](https://static.igem.org/mediawiki/2021/b/b6/T--MSP-Maastricht--digestion1.png)

For the Enzyme:
Keep always on ice
Last component to add
Mix components by pipetting mixture up and down - flick reaction tube
In general - recommended 5-10units of enzyme per ug DNA & 10-20 units for genomic DNA in a 1 hour digest

DNA: Free of contaminants.

Buffer: Use at 1x concentration.

Reaction volume:
![](https://static.igem.org/mediawiki/2021/4/4d/T--MSP-Maastricht--digestion2.png)

## References

+ Machado, L., Magnusson, M., Paul, N. A., Kinley, R., de Nys, R., & Tomkins, N. (2016).
  Identification of Bioactives from the Red Seaweed Asparagopsis taxiformis that
  Promote Anti-methanogenic Activity in vitro. Journal of Applied Phycology, 28(5),
  3117–3126. doi:10.1007/s10811-016-0830-7
+ Roque, B. M., Brooke, C. G., Ladau, J., Polley, T., Marsh, L. J., Najafi, N., ... & Hess, M. (2019). Effect of the macroalgae Asparagopsis taxiformis on methane production and rumen microbiome assemblage. Animal Microbiome, 1(1), 1-14.
+ Thapa, H. R., & Agarwal, V. (2021). Obligate Brominating Enzymes Underlie Bromoform
  Production by Marine Cyanobacteria. Journal of Phycology.
+ Thapa, H. R., Lin, Z., Yi, D., Smith, J. E., Schmidt, E. W., & Agarwal, V. (2020). Genetic and
  Biochemical Reconstitution of Bromoform Biosynthesis in Asparagopsis Lends
  insights into Seaweed Reactive Oxygen Species Enzymology. ACS Chemical Biology,
  15(6), 1662-1670.
+ Zhu, P., Li, D., Yang, Q., Su, P., Wang, H., Heimann, K., & Zhang, W. (2021). Commercial
  Cultivation, Industrial Application, and Potential Halocarbon Biosynthesis Pathway
  of Asparagopsis sp. Algal Research, 56, 102319.
  https://doi.org/10.1016/j.algal.2021.102319

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

# Team:MSP-Maastricht/Results

![](https://static.igem.org/mediawiki/2021/b/ba/T--MSP-Maastricht--logoBrown.png)

* [Home](https://2021.igem.org/Team%3AMSP-Maastricht)

* Team
  + [Team](https://2021.igem.org/Team%3AMSP-Maastricht/Team)
  + [Attributions](https://2021.igem.org/Team%3AMSP-Maastricht/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AMSP-Maastricht/Collaborations)

* Project
  + [Contribution](https://2021.igem.org/Team%3AMSP-Maastricht/Contribution)
  + [Description](https://2021.igem.org/Team%3AMSP-Maastricht/Description)
  + [Engineering](https://2021.igem.org/Team%3AMSP-Maastricht/Engineering)
  + [Experiments](https://2021.igem.org/Team%3AMSP-Maastricht/Experiments)
  + [Notebook](https://2021.igem.org/Team%3AMSP-Maastricht/Notebook)
  + [Proof Of Concept](https://2021.igem.org/Team%3AMSP-Maastricht/Proof_Of_Concept)
  + [Proposed Implementation](https://2021.igem.org/Team%3AMSP-Maastricht/Implementation)
  + [Model](https://2021.igem.org/Team%3AMSP-Maastricht/Model)
  + [Results](https://2021.igem.org/Team%3AMSP-Maastricht/Results)

* [Parts](https://2021.igem.org/Team%3AMSP-Maastricht/Parts)

* [Safety](https://2021.igem.org/Team%3AMSP-Maastricht/Safety)

* [Human Practices](https://2021.igem.org/Team%3AMSP-Maastricht/Human_Practices)
  + [Human Practices](https://2021.igem.org/Team%3AMSP-Maastricht/Human_Practices)
  + [Collaborations](https://2021.igem.org/Team%3AMSP-Maastricht/Collaborations)

* [Education & Communication](https://2021.igem.org/Team%3AMSP-Maastricht/Communication)

* Awards
  + [Education](https://2021.igem.org/Team%3AMSP-Maastricht/Education)
  + [Entrepreneurship](https://2021.igem.org/Team%3AMSP-Maastricht/Entrepreneurship)
  + [Hardware](https://2021.igem.org/Team%3AMSP-Maastricht/Hardware)
  + [Inclusivity](https://2021.igem.org/Team%3AMSP-Maastricht/Inclusivity)
  + [Model](https://2021.igem.org/Team%3AMSP-Maastricht/Model)
* [Documentary](https://2021.igem.org/wiki/index.php?title=Team:MSP-Maastricht/Documentary)

# Results

The project consisted of 2 sections completed consecutively. The first being the Drylab
section that was
completed at the beginning of the year when Covid-19 regulations were heavily implemented. It consisted of
the creation of the spacer system as well as a metabolic model. Once able to access the lab, the team
continued on with the Wetlab work which included testing the spacer system using PCR, Gels, and
Sequencing, and completing the ruminal simulation(Gut in Glass).

## Drylab

### Spacer System

The Spacer system is part of our [contribution](https://2021.igem.org/Team%3AMSP-Maastricht/Contribution)
to future iGEM teams and represents a buffer system for the targeted gene.

The idea of the Spacer system is that we can create new gBlocks using PCR with the single genes. These newly obtained constructs are then able to be combined with one another. This allows for varying combinations of any gene sequences that have the correct overhangs complementary to each other. The results of testing the Spacer in SnapGene cloning algorithms showed the full integration of the developed Forward and Back spacers into the tested genes. The conducted simulation of Gibson assembly with specific primers that would anneal to the spacers run successfully.

In the wetlab, PCR's were run successfully, and combinations were achieved using Gibson assembly. The assembled genes, which were inserted into a plasmid, were transformed into E.coli bacteria, grown on agar plates using Kanamycin as a selective condition, and re-plated once growth was detected. After successful mini-preps, both agarose gels and sequencing results confirmed the efficacy of the spacer system. Therefore, this new part can be further used to assess varying gene combinations with basic lab procedures, independent from outside sources.

![](https://static.igem.org/mediawiki/2021/4/46/T--MSP-Maastricht--Proof_1.jpg)
![](https://static.igem.org/mediawiki/2021/e/ee/T--MSP-Maastricht--Proof_2.jpg)

### Model

From our metabolic modeling efforts, we find that E.coli is able to produce 36.04 mmol bromoform/gDW/hour,
which is physiologically feasible. The limiting factor on bromoform production is the available amount of
hydrogen peroxide in the cell, which is required to produce the main compound needed for bromoform
production - HOBr.

With this information, we can create an estimate of how much bromide we need to include in our feed additive
to generate an amount of bromoform that is sufficient for the elimination of methanogenesis in cow rumen.

`
[Modelling Page](https://2021.igem.org/Team%3AMSP-Maastricht/Modelling)

## Wetlab

### Successful Transformation and Growth

Plasmids with sets of triplex, duplex, and single genes were transformed, and multiple colonies were selected
for each of them. To verify the authenticity of the genetic composition of the colonies with the predicted,
sequences were successfully extracted and checked for the right genes using a miniprep procedure and visualization via
gel electrophoresis. According to the results, only colonies with single and duplex genes gave successful
growth, which was predictable and did not interfere with the flow of further tests.

![](https://static.igem.org/mediawiki/2021/d/da/T--MSP-Maastricht--agar_plates.jpg)

### PCR

Since the Spacer System was used in each gene construct, the g-blocks could be generated in one PCR cycle
with all the corresponding overhangs necessary for their assembly. In our case, small adjustments to the
temperature mode of the procedure were introduced due to the temperature sensitivity of the primers that
anneal with the Back spacer, which contains the overhang of the Forward spacer: if the temperature is too
high, reverse binding of the primer may take place. This means that the primer would anneal on the overhang
side which is complementary to the Forward Spacer rather than the intended location on the Back Spacer. With
these considerations in place, the spacers proved to be effective in the generation of overhangs necessary
for Gibson assembly.

![](https://static.igem.org/mediawiki/2021/d/d6/T--MSP-Maastricht--cool_pour.jpeg)

### Gels

![](https://static.igem.org/mediawiki/2021/7/71/T--MSP-Maastricht--gels.jpg)
With the help of regularly conducted gels, the presence of single and duplex genes in samples was constantly
monitored. To facilitate gene restriction digestion, the restriction sites were placed in the Spacer system
Specific unique enzyme cutters were used to prevent any accidental nicks in the gene, as these
would result in "noise" in visual representation. A similar procedure was used with the duplex genes, while single
gene constructs were shown to be transformed successfully, that is, to have correct band location after
restriction digestion. To ensure that constructed genes are present and perform as desired, the
genotypes of different colonies underwent sequencing. Two out of six single gene constructs (MBB1 and
CcVHPO1) were able to match up with complete accuracy with the constructs designed, so they were selected for
further protein expression and potential testing in the Gut in Glass ruminal simulation.

Further protein extraction did not provide us with useful insight about the fact of the presence of
bromoform, and we intend to conduct further analyses to confirm the fact of its creation, with the
expectation of results similar to the data of the metabolic model output.

### Gut in Glass

Overall, the control simulation produced much more gas than the bromoform treated simulation. Yet there were different steepness’ of the graphs over time, as can be seen in charts 1 and 2.

![](https://static.igem.org/mediawiki/2021/c/ca/T--MSP-Maastricht--results_graph_1.png)

Chart 1 Amounts of gas produced over a time period of 26 hours and 40 minutes. Green lines
represent
removal of old feed and addition of new feed.

![](https://static.igem.org/mediawiki/2021/3/39/T--MSP-Maastricht--results_graph_2.png)

Chart 2 Amounts of gas produced over a time period of 26 hours and 40 minutes. Green lines
represent
removal of old feed and addition of new feed.

The red line represents an invalid reading. Blue lines indicate the addition of 20uL of Bromoform.

Discussion:
The bromoform treatment did increase its production on the second day, which may be due to the initial
bromoform being used up.
The stagnant gas production overnight can be explained by the lack of newly added saliva buffer and
feed. The production increased once again on the next day, as a new buffer and feed was added, therefore
confirming that the buffer and feed are necessary additions to the simulation.

This experiment gives us a great basis to work with in future experiments, as it is confirmed as
anaerobic, has a way of measuring and trapping produced gas, and allows for control over most variables.
In future experiments, fatty acid concentrations would be measured, along specific methane gas content
measurements. These experiments will use the same setup as the one described here. For further reading,
see [here](https://2021.igem.org/Team%3AMSP-Maastricht/Modelling).

![](https://static.igem.org/mediawiki/2021/9/99/T--MSP-Maastricht--Footer-full-logo.png)

## Sponsors

![Maastricht University logo](https://static.igem.org/mediawiki/2021/c/cf/T--MSP-Maastricht--Footer-UM-logo.png)

![Integrated DNA Technologies logo](https://static.igem.org/mediawiki/2021/8/88/T--MSP-Maastricht--Footer-IDT-logo.png)

![SnapGene logo](https://static.igem.org/mediawiki/2021/a/a7/T--MSP-Maastricht--Footer-SnapGene-logo.png)

![Brightlands Venture Partners logo](https://static.igem.org/mediawiki/2021/e/e7/T--MSP-Maastricht--Footer-Brightlands-logo.png)

![GenScript logo](https://static.igem.org/mediawiki/2021/f/fc/T--MSP-Maastricht--Footer-GenScript-logo.png)

![HadeCo logo](https://static.igem.org/mediawiki/2021/f/fc/T--MSP-Maastricht--Footer-imgB-logo-hadeco2.jpeg)

Instagram: [msp\_igem](https://www.instagram.com/msp_igem/)
Linked-in: [iGEM MSP-Maastricht](https://www.linkedin.com/company/msp-igem-maastricht/)
Youtube: [Geneducation](https://www.youtube.com/channel/UCpZQvRTiQNvqz9XcqDGK9pA/featured)
Facebook: [MSP iGEM](https://www.facebook.com/MSPIGEM)
Tik-tok: msp\_igem