iGEM 2021 — Team NOVA_LxPortugal

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# Team:NOVA LxPortugal/Notebook

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Project

[Overview](https://2021.igem.org/Team%3ANOVA_LxPortugal/Description)

[Safety](https://2021.igem.org/Team%3ANOVA_LxPortugal/Safety)

[Engineering Success](https://2021.igem.org/Team%3ANOVA_LxPortugal/Engineering)

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

Lab

[Wet Lab](https://2021.igem.org/Team%3ANOVA_LxPortugal/Experiments)

[Parts](https://2021.igem.org/Team%3ANOVA_LxPortugal/Parts)

[Project Modelling](https://2021.igem.org/Team%3ANOVA_LxPortugal/Model)

Future

[Proposed Implementation](https://2021.igem.org/Team%3ANOVA_LxPortugal/Implementation)

[Contributions](https://2021.igem.org/Team%3ANOVA_LxPortugal/Contribution)

Human Practices

[Integrated Human Practices](https://2021.igem.org/Team%3ANOVA_LxPortugal/Human_Practices)

[Education & Communication](https://2021.igem.org/Team%3ANOVA_LxPortugal/Communication)

[Collaborations](https://2021.igem.org/Team%3ANOVA_LxPortugal/Collaborations)

[Multimedia](https://2021.igem.org/Team%3ANOVA_LxPortugal/Photography)

Team

[Team Members](https://2021.igem.org/Team%3ANOVA_LxPortugal/Team)

[Attributions](https://2021.igem.org/Team%3ANOVA_LxPortugal/Attributions)

[Home](https://2021.igem.org/Team%3ANOVA_LxPortugal)

# Notebook

Index

## March

### 1st week (March 16th to March 30th)

This is the first time we heard about iGEM. Two weeks before the first official meeting we were informed of the possibility to participate in this event. It was also raised the possibility of having a meeting with students that participated in the previous year to share their experience and what we could expect if we embark on this adventure.

### 2nd week (March 30th to April 5th)

So, on the 30th of March 2021, the first meeting occurred, where we for the first time informed about our interest in participating in iGEM. It was also at this meeting that we got together with previous students who participated in the previous year, to answer some doubts we had about participation in iGEM and to get some knowledge of the experience that the two students had during their participation. Additionally, the brainstorming about the suitable theme for development has begun.

## April

### 3rd week (April 6th to April 12th)

In this meeting, several ideas for the iGEM project were discussed. Our advisor has proposed the repartition of students into small groups for further research on the proposed ideas and then share their findings the following week. We also created our official email to address further needs.

### 4th week (April 13th to April 19th)

There were many ideas presented in this meeting:

 1) The removal of plastics from the ocean water through a vacuum cleaner like machine;

 2) The substitution of the Haber-Bosch process;

 3) Trying to combat a plant plague, preferably, a portuguese associated one was also an idea in discussion.

Afterwards, we made the following groups to come up with solid and attractive presentations for further voting. Groups composition: Haber-Bosch (Guilherme, Ana, Fábio, André and Bárbara), Microplastic Degradation (Olga, Rodrigo, Vitor, Rita, Henrique, Alexandre) and Plant Plague (Francisco, David, Marta, Fredi and Diogo).

### 5th week (April 20th to April 26th)

We proceeded with the presentations. All presentations were catchy, informative and compelling. It will be a tough decision to make.

### 6th week (April 27th to May 3rd)

After a whole week of deliberation, the most voted project proposal, the bacterial degradation of microplastics. We have decided our theme! Let’s get to work! For logistic matters team members were divided into 3 (bibliographic) research teams, proposed to address distinct aspects of the project: (i) microplastic composition; (ii) microorganisms and/or enzymes involved in microplastic degradation; and (iii) technologies involved in the collection and/or treatment of microplastics.

Now the fun part has begun, the search to make our project come true. Baby steps!

## May

### 7th week (May 4th to May 13th)

We got to share our first finding with each other. The following subtopics were focused:

 1) Microplastics Composition;

 2) Microorganisms/Enzyme capable of Degrading Plastics ;

 3) Technologies for harvesting and processing microplastics .

This meeting was also extremely exciting since we had to proceed with the election of the Student Leader. First thing first, we started with nominations and applications, and the following week was to vote on who we though would suit the best in this position.

### 8th week (May 14th to May 17th)

This week was only about good news, we chose our team leader – Guilherme Alves. Furthermore, our team had a little member growth. With the join of new members, we made a quick tour on what have been discussed so far and proceeded with the exposition of our analysis: microorganisms used to degrade the microplastics, such as *E.coli*, *Bacillus* and *Pseudomonas*.

It was suggested by our supervisor to focus on some microorganisms like *Pseudomonas* and to search for different metabolic pathways related to degradation of microplastics.

### 9th week (May 18th to May 27th)

We manage to come down to two microorganisms possible for our project, now only left to choose which one is better. To do so – we need a little bit of research. The following task consisted in gathering the molecular information on *Bacillus* and *Pseudomonas*.

However, the project is not only the research we also did a little brainstorming on the next steps of the project (possible application of this project in beach cleaners) and some doubts were clarified. Our meeting started to be more lively and fuller, like this:

![](https://static.igem.org/mediawiki/2021/1/18/T--NOVA_LxPortugal--noteFIG1.jpeg)

### 10th week (May 28th to May 30th)

After having our project and our microorganism chosen, *B. subtilis*, we started to collect the information on laboratorial procedures and the products needed for our research. Well, smells like another task is coming up.

## June

### 11th week (June 1st to June 17th)

The meeting started with the sequence analysis of our bacteria. after we proceeded with the order of the peptides and sequence that will possibly be used in experimental work. Really exciting and important task.

The new assignments were distributed to groups, as listed below:

  1) Mutant enzyme sequence: Ana, Francisco, Vítor

  2) Promoter and signal sequence: Guilherme, Marta, César, Fábio, Alexandre

  3) List of protocols and reagents: Rodrigo, Bárbara, David, Fredi, André

  4) Plasmid Cloning and Terminators: Pedro, Olga, Henrique

At this point we have many things to do, no time for slaking.

### 12th week (June 18th to June 21st)

At this point, we are already choosing our project name, we came up with idea of P(L)AST the ocean cleaner. Cool right, it is like a band-aid you put on your wound to protect, the same as with our project, that is meant to protect the oceans.

During previous week, we conducted an interview to Paula Sobral where she gave her specialist opinion. She also proposed and mentioned the necessity of biosensors in this area, in order to identify microplastic, which made us wonder about our original idea.

Next, groups were made:

  1) **Wet Lab:** Ana Ferreira, André Piteira, Bárbara Fernandes, César Duarte, David Cortiço, Fábio Queiróz, Francisco Sobral, Fredi Brigham, Guilherme Alves, Henrique Silva, Marta Cação, Olga Chervonovska, Rodrigo Barriga Alves e Vitor Mordido

  2) **Engineering:** Alexandre Coelho, André Piteira, César Duarte, Guilherme Alves, Marta Cação

  3) **Sponsors & Financial Analysis:** Diogo Torres, Olga Chervonovska, Inês Ventura

  4) **Computational/Modelling:** Alexandre Coelho, Fábio Queiróz, Francisco Sobral, Fredi Brigham, Henrique Silva, Rodrigo Barriga Alves

  5) **Human Practices/Impact:** Ana Ferreira, Bárbara Fernandes, Pedro Nunes, Rodrigo Barriga Alves, Vitor Mordido, Inês Ventura

  6) **Communication:** David Cortiço, Diogo Torres, Pedro Nunes, Rita Teixeira, Inês Ventura, Olga Chervonovska, Rodrigo Barriga Alves

### 13th week (June 22nd to June 29th)

After Paula’s Sobral interview the group was divided between biosensor and degrading technology. After some voting and discussion, we finally managed to come up with the final decision! Which one is it going to be?

We choose our original idea the degrading technology. Well, they always say, original is always better! We thought so too!

### 14th week (June 30th to July 6th)

Since we already have the groups established each has now a specific task to do.

  1) **Informatics/Modelling:** this week was about organization and task distribution.

  2) **Wet Lab:** MHETase strategies (Marta, André, Henrique, Fábio, Bárbara). We ordered our material, however there were delay and problems in the customs, we wonder when our plasmids are going to come so we can start our work. We have this idea of implementing the *Idionella* PETase and MHETase into *Bacillus*, by doing this we hope to power up *Bacillus* and make it more efficient in microplastic degradation.

  3) **Engineering:** state point and discussion of project, how we can implement it, what are the best ideas and ways of doing it.

  4) **Finances:** have already started doing powerpoint for companies and gathering possible sponsors and contacts. As soon as the powerpoint is finished we will send it to the companies.

  5) **Communication:** this one had the most work to do. Starting with the promotional video. We choose to make video a little bit cartoonish. Everyone loves watching cartoons, right? Not only we could attract older audience but also the younger one! Also we discussed several ideas for Educational purposes: the idea of “Synthetic Biology Event” – mainly directed to university students; environmental side – directed to primary or secondary classes; taking part in beach cleaning activities; solidary events; example: bottle lids. The most talented ones are also starting to create our logo. So excited about the outcome!

  6) **Human Practices:** they also did not waste time, started right away gathering the information on contacts for collaboration, related projects and further collaboration possibilities.

## July

### 15th week (July 7th to July 12th)

We conducted different meetings during the week, and present the highlight on general meeting, so everyone is aware of all the work.

**Engineering:** it was presented which type of microplastic is most problematic - particularly PET and PE. The reason behind it is that most microplastics were found in sediments, mainly PET - because it has a higher density than water. Additionally, a reaction caused by UV radiation leads to PE precipitation.

It was also discussed microplastics separation strategies, it was proposed a two steps purification protocol: one at the local of collection, to limit sediments quantity transferred; another later to obtain a more purified and enriched sample in microplastics - since some sediments and microplastics might present similar characteristics.

For a location to implement the system, it was first suggested ETARs (Estações de Tratamento de Águas Residuais - residual water treatment facilities). Another possibility for an application site would be piscatory regions, as the fishing nets are one of the causes of microplastics in water.

For the collection of sediments and water it was suggested the use of dredgers, a boat designed for dredging harbors. However, further research are required in terms of the legislation and companies interested in it.

**Communication, Sponsors, and Human Practices:** In the meeting with the ITQB communication office, it was considered the possibility of organizing a workshop for ITQB, IGC, and iMM about the reduction of plastic consumption in the laboratories, as well as, implementing plastic recycling in ITQB. They will also provide help with institutional contacts (such as the Pavilhão do Conhecimento, Oceanário, and Aquário Vasco da Gama) and promote communication with the city councils.

Regarding the possibility of making a construction with collected plastics, it was suggested sending an email to all ITQB personnel – all ideas are welcome, the more the merrier. Another option was doing it as a project with different schools.

The communication group has also finished the presentation of the project, and the guide for the promotion video has been written – next stop: execution and drawing!

![](https://static.igem.org/mediawiki/2021/7/71/T--NOVA_LxPortugal--noteFIG8.jpeg)

**Wet Lab:** In the previous meeting, work was divided into the search for more appropriate protocols, define the MHETase strategy, and identify the best mutant enzymes.

Our first first results and our lab WORK! We started with the performance of a PCR to amplify the genes with the ordered primers and the amplification quality was verified. So, later they can be inserted into the plasmids.

**Modelling:** Currently, it is being researched multiple *Bacillus* models, for comparison, to identify which would be the best model to use.

Who said research work is easy? We need to search and compare different models and find the most suitable one! Chop chop, time wait for no one!

### 16th week (July 13th to July 19th)

![](https://static.igem.org/mediawiki/2021/3/3b/T--NOVA_LxPortugal--noteFIG9.jpeg)

**Wet Lab:** it was presented the best mutant enzymes. We found that relative to PETase mutation there are a lot of mutants, to be more precisely there are 13 of them which lead to an increase of activity. There is also a linker which binds PETase and MHETase also increasing their activity. It is a promising start!

Also, good news, our plasmid kit directly from Headquarters is finally here! How let’s get our hand dirty! If you know what we mean!

Great news! Our dear friend Beatriz Almeida has finished drawing the promotional video! Now we are going to do a voice over! Our lovely Inês Ventura was chosen to be the narrator, unanimously. And it is done!

### 17th week (July 27th to August 2nd)

**Communication:** A weekly news system regarding microplastics was created to keep the team updated on current events concerning the topic. Also, we created our social media account (Instagram) for this as an outreach strategy. We also discussed the enrollment on the Latin American encounter. Our team members were challenged to make a small video explaining why they wanted to join the team, how they know about iGEM, and their interests.

**Sponsors:** Details regarding the proposal pitch were discussed and adjusted, as were optimal contact dates to potential sponsors. A crowdfunding project (GoFundMe®) was created for the team in order to collect the donations.

**Wet Lab:** We had a little trouble, since the plasmid was nonfunctioning, so we managed to contact the Uppsala team to help solve the problem. However, it did not stop us from doing another essential task such as elaboration of a list of programmed activities (cloning strategies, tests involving signal peptides, PET degradation assays).

## August

### 18th week (August 3rd to August 9th)

**Communication:** It started by defining the strategy to promote GoFund Me, using ITQB communication services and later sending emails to all ITQB members.

**Wet Lab:** Plasmids have not arrived yet, however the design strategy, protocols and mutants are defined, André compiled the work that has been done so far and the work plan (English).

**Human Practices:** document compiled with contacts, document with structured events (powerpoint), beach cleaning event (with town hall, with NAS, with scouts) were made. Our team members are so organized!

**Informatics Modelling:** checking if *Bacillus* can degrade ethylene glycol, which enzymes we need to add. The main focus is, however, ethylene glycol since it is the energy source of system.

We started organizing the Wiki and we are participating in LATAM, so we are looking for volunteers to do the presentation!

### 19th week (August 10th to August 16th)

Each group discussed what had been done during that week, progress, and the difficulties they had encountered. We are taking shifts to go to the laboratory. Everyone is eager to go!

### 20th week (August 17th to August 23rd)

Starting with good news, we have our LATAM speakers: André Luís, Guilherme Alves, Olga Chervonovska, Henrique Pereira

**Wet Lab:** Since there is a delay in laboratory work, research on following matters was requested: conditions to be used in the bioreactor (tolerance of bacteria, preferential conditions, alternative carbon/energy sources).

**Modelling:** already has the idea of the pathway degradation of PET to generate energy through plastic. They are undecided whether to use glycolate, but apparently there may be other alternatives, and the first way is not easily executed, they are studying other possibilities. The model does not have a shuttle that is required. Further research is needed.

### 21st week (August 24th to August 31st)

We are preparing for LATAM! The presentation is on the 28th - this is the main event this week. Everyone is excited. While our volunteers are dedicated to this presentation, the rest of the team is working hard on further research.

Engineering is looking for ways to conciliate our degrading bacteria with ETAR’s bioreactors. Either with the insertion of a new tank only meant for microplastic treatment or combining other tanks with bacteria. Pretty genius, right?

**Human Practices:** this part of the team is preparing Conferência do Mar. Event where we invite other iGEM teams and specialists to talk about their project and work.

![](https://static.igem.org/mediawiki/2021/f/f5/T--NOVA_LxPortugal--noteFIG10.jpeg)

## September

### 22nd week (September 1st to September 6th)

Our university has a lot of events coming up, and we are thinking of making our presence there. There are a lot of logistics behind every little event, our hardworking bees haven´t stopped yet.

### 23rd week (September 7th to September 14th)

We received a proposal from a Mexican team for collaboration. Fingers crossed; we hope everything will be fine.

We are having problems with our plasmids, so we need to change our strategies, we need to use different sets of primers. We are confident in our work, we know everything will be fine.

### 24th week (September 15th to September 20th)

Everyone is so busy with their master thesis. However, there is always time for iGEM.
Wet Lab proceeded with the creation of an experimental protocol for tag exportation efficiency in *Bacillus Subtilis*, alternative protocols were set to be discussed further.
HumanPractises planned for upcoming public events.

Creation of drafts with information for the teams Wiki page were set to be created for each group. It has begun. One month until official wiki FREEZE.

### 25th week (September 21st to September 30th)

We are working on parts for Wet lab. Our biochemist Barbara is responsible for this task, it is important since this work will be left for following iGEM generations get inspired and guided.
Another good news, we are having success in the Lab! Check this out:

![](https://static.igem.org/mediawiki/2021/5/51/T--NOVA_LxPortugal--noteFIG11.jpeg)

## October

### 26th week (October 1st to October 8th)

It is now 21 days left for wiki freeze. Our team started to fill what is missing. We are not panicking. Yet. We are just stressed.

### 27th week (October 9th to October 16th)

This week our three volunteers - André, José and Olga - went to primary school; this visit meant to sensitize on the issue of microplastics and the excessive use of plastics. Well, what can we say, the children were amazing, really smart and really curious. A lot of them have good daily habits so we really felt that our purpose was understood. It was such a pleasure!

![](https://static.igem.org/mediawiki/2021/e/e6/T--NOVA_LxPortugal--FIGtalk2.jpeg)
![](https://static.igem.org/mediawiki/2021/a/aa/T--NOVA_LxPortugal--FIGtalk4.jpeg)

### 28th week (October 17th to October 21st)

We are really working on Wiki, there were some delays, but this won´t stop us from finishing our wiki. Some sleepless nights won’t harm anyone.

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# Team:NOVA LxPortugal/Engineering

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Project

[Overview](https://2021.igem.org/Team%3ANOVA_LxPortugal/Description)

[Safety](https://2021.igem.org/Team%3ANOVA_LxPortugal/Safety)

[Engineering Success](https://2021.igem.org/Team%3ANOVA_LxPortugal/Engineering)

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

Lab

[Wet Lab](https://2021.igem.org/Team%3ANOVA_LxPortugal/Experiments)

[Parts](https://2021.igem.org/Team%3ANOVA_LxPortugal/Parts)

[Project Modelling](https://2021.igem.org/Team%3ANOVA_LxPortugal/Model)

Future

[Proposed Implementation](https://2021.igem.org/Team%3ANOVA_LxPortugal/Implementation)

[Contributions](https://2021.igem.org/Team%3ANOVA_LxPortugal/Contribution)

Human Practices

[Integrated Human Practices](https://2021.igem.org/Team%3ANOVA_LxPortugal/Human_Practices)

[Education & Communication](https://2021.igem.org/Team%3ANOVA_LxPortugal/Communication)

[Collaborations](https://2021.igem.org/Team%3ANOVA_LxPortugal/Collaborations)

[Multimedia](https://2021.igem.org/Team%3ANOVA_LxPortugal/Photography)

Team

[Team Members](https://2021.igem.org/Team%3ANOVA_LxPortugal/Team)

[Attributions](https://2021.igem.org/Team%3ANOVA_LxPortugal/Attributions)

[Home](https://2021.igem.org/Team%3ANOVA_LxPortugal)

# Engineering Success

Index

## Computational Model Improvement: 2+ Iterations of the Engineering Success Cycle

Here we present our engineering success whose main objective was to develop a genome-scale metabolic model of *Bacillus subtilis* able to support simulations and optimizations using PET and PE - the major constituents of microplastics - as the carbon sources to grow.

### RESEARCH

Several genome-scale metabolic models of *B. subtilis* already exist and could be used as the origin of our project. However, none of them included the PET or PE degradation pathways, so these pathways had to be inserted as a part of our project regardless of the chosen model. Additionally, the majority of the available models were not in the ideal SBML format, hampering the application of the required modifications and further simulations. Some of the candidate models to be converted into SMBL were too time intensive for the span of the project and were discarded. Therefore, a small set of possible models to be converted to SBML were then analyzed. We finally opted for the *i*Bsu1103 model [1], which was adapted to be the basis of our work, despite being one of the oldest models available, it was manually annotated and covers the majority of the metabolism, only differing slightly in size and complexity from more recent models.

### IMAGINE

Our main goal was to incorporate all of the reactions found in the PET and PE degradation pathways into our model and use it to simulate the consumption of these polymers as the only sources of carbon for our modified *B. subtilis* strain. Thus, supporting the simulations and optimizations to find an optimum genotype which would allow our GMO to survive and possibly thrive in PET and/or PE rich environments. We also intend, in later stages of the project, to use the optimized model for the production of innocuous (e.g. ethanol) or possibly high/added value compounds (e.g. surfactin) from these carbon sources. Optimization tools can be used to identify the best suited candidates for metabolic engineering (i.e upregulation, downregulation and knockout) by maximizing ethanol production whilst maintaining biomass growth in an environment containing PET or PE as a carbon source. This project must be developed in tandem with the WetLab group in hopes of aiding in the metabolic target discovery process.

Nevertheless, to achieve this stage, a functional model integrating PET and PE degradation pathways is required.

### DESIGN

Once the most suitable model was chosen (*i*Bsu1103) as a starting point, new metabolites and reactions specific to our project had to be added. After understanding the degradation pathway of PET, we realized that we could link the metabolite Ethylene Glycol (EG) to the cell's metabolism as an alternative carbon source. For this reason, EG and the respective reactions that link it to the cell's endogenous metabolism were first added.

### TEST

After these inputs, we used the software Optflux (version 3.4.0) to run simulations with the cell using EG as the only carbon source. Unfortunately, the biomass growth rate obtained during these simulations was null. Since biomass growth rate value determines the cell's efficiency and likelihood of survival, the results indicate that either cell growth is not feasible using EG as the sole carbon source or there is an error in the metabolic model.

### LEARN

A drain is a hypothetical reaction that represents the consumption or production of metabolites by the system. This can be done by transforming a non-existent hypothetical metabolite (cpd99999\_b) into an external metabolite (cpd99999\_e). By adding this reaction to the SBML model as presented in Figure 1, we were able to run simulations and get the result seen in Figure 2.

![](https://static.igem.org/mediawiki/2021/7/75/T--NOVA_LxPortugal--ESfig1.png)

**Figure 1**: Required reaction added to the SBML file of model *i*Bsu1103 highlighted in green.

![](https://static.igem.org/mediawiki/2021/c/c6/T--NOVA_LxPortugal--Esuccess_fig2.jpeg)

**Figure 2**: WT simulation using model *i*Bsu1103 with the addition of metabolite Ethylene Glycol and respective reactions, as well as Environment Conditions adding PET to the media instead of Glucose.

As depicted in Figure 2, the biomass growth rate was 140.71 h-1 in our simulations which possibly validates cell growth when using EG as the sole carbon source.

### IMPROVE

We decided to first add the entire PET degradation pathway and lastly the PE degradation pathway. Once again, the biomass growth rate was null when using PET as the only carbon source. All reactions until EG enters the cell are extracellular, as well as every metabolite involved in those reactions. Therefore likely EG, it was also necessary to add drains for all those metabolites, representing their presence on the surrounding medium.

### TEST

After adding these reactions, we were able to simulate cell growth when using PET as the only carbon source (Figure 3). In this simulation, contrary to the one with EG, PET intake was limited to better represent real life conditions, which is why there is a smaller growth rate (0.90481 h-1).

![](https://static.igem.org/mediawiki/2021/b/bc/T--NOVA_LxPortugal--ESfig3.png)

**Figure 3**: WT simulation using model *i*Bsu1103 using PET as the only carbon source in the medium

Since, alongside PET, PE is the major constituent of microplastics, we wanted to perform simulations where both PET and PE are used as the carbon sources by our cells. Therefore, and learning from the previous design problems, the PE degradation pathway was inserted into the model as well. However, simulations with only PE as carbon source showed null growth rate. These results are further explained in the [Project Modelling](https://2021.igem.org/Team%3ANOVA_LxPortugal/Model) page.

### New round of optimization

Our model is still incomplete as it does not include the degradation pathway of the TPA metabolite, leading to an overproduction and secretion in the simulation results. Consequently, the incorporation of the TPA degradation pathway must be the subject of our third iteration on the Engineering Cycle. We plan on adding this pathway to our model and re-test it so we could determine if any further improvements are required or if the resulting model is already ready to support reliable simulations and optimizations.
In Figure 4 we have represented our Imagine-Design-Test-Learn-Improve work cycles used for the construction of our model so far. In the future, similar cycles should be used for the next engineering problems.

![](https://static.igem.org/mediawiki/2021/d/d1/T--NOVA_LxPortugal--last_image_engineering_1.png)
![](https://static.igem.org/mediawiki/2021/7/74/T--NOVA_LxPortugal--last_image_engineering_2.png)

**Figure 4**: Schematic of our work cycles.

## References

1. Henry, C. S., Zinner, J. F., Cohoon, M. P., & Stevens, R. L. (2009). *i*Bsu1103: A new genome-scale metabolic model of Bacillus subtilis based on SEED annotations. Genome Biology, 10(6), R69. https://doi.org/10.1186/gb-2009-10-6-r69

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# Team:NOVA LxPortugal/Experiments

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# Wet Lab

Index

## Strain Selection

There are several species of bacteria capable of degrading microplastics, mainly those that form a biofilm on the surface of polyethylene [1]. The two of the genera that have shown more promising results in the biological degradation of both PET and PE are the *Bacillus* and *Pseudomonas*. *Pseudomonas spp.* alone were reported to degrade 35 different plastic types, including most of the mass-produced polymers [2,3] with the species *Pseudomonas aeruginosa* being one of the most efficient PET degradation bacteria [1]. However, in this project, the *Bacillus subtilis* species was chosen, which by itself has a low PET degradation activity but, due its ease of transformation it allows the expression of the efficient PETase and MHETase enzymes of *Ideonella sakaiensis*. Moreover, the *B. subtilis* secretion system is well-known and was already studied and industrially implemented to secrete a wide variety of enzymes. Finally, its ability to use PE as a carbon source for its growth will lower the culture maintenance costs.

## Materials and Methods

### Cloning Procedure

Codon-optimized genes for expression in *Bacillus subtilis* were obtained from IDT (Iowa, USA). The genes used in this study were amplified by polymerase chain reaction (PCR) using Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Waltham, USA) in a LifeECO Thermal Cycler (Bioer Technology, Zhejiang, China). All primers were purchased from IDT. DNA fragments were purified using DNA Clean and Concentrator DNA Kit (Zymo Research, Irvine, USA). Plasmids were extracted using Plasmid Miniprep kit (Zymo Research). Clones were obtained by performing FastCloning [4] and transformed by heat-shock in chemically competent *E. coli* NEB 5-alpha cells (New England BioLabs, Massachusetts, USA). The success of ligation was visually checked by picking white colonies instead of pink ones – due to the substitution of the gene RFP in positive clones – and further confirmed by sequencing (StabVida, Lisbon, Portugal). Protocols were performed in accordance with manufacturer’s instructions.

For long-term storage, glycerol was added to a final concentration of 30 % (v/v) to overnight cultures in selective media and kept in a -80 °C freezer.

### Plasmid Construction

Our plasmid constructs use the pBS1C and pBS2E plasmids as vectors to express our target gene sequences. We used two different chassis, each containing one of our insert sequences, featuring either PETase or MHETase sequences, so that we could select the cell strains that contained both PETase and MHETase, using the two different antibiotic resistances conferred by these plasmids. Both of our insert sequences feature a similar design, only changing the target protein sequence.

Analysing in detail our insert sequences, they start with the P43 promoter sequence - a strong constitutive promoter already validated for gene expression in *Bacillus* systems which activates the transcription during exponential and lag phases of growth. Followed by the ribosome binding site (RBS), this sequence in bacterial mRNA ensures the translation of proteins in *Bacillus subtilis*. Then, the native signal peptide of each enzyme, present in our design, ensures that both enzymes, PETase and MHETase, are properly secreted to the extracellular matrix. In the case of PETase an additional signal peptide was tested, the SPamy. Located between the signal peptide sequence and the T7 terminator is the target protein sequence which will be further detailed below for both our enzymes. Our insert sequence design is then finalized by a rho-independent T7 terminator (T7Te) which encodes for an RNA sequence that can form a stable stem-loop structure followed by a run of six uridylate residues thus allowing for an efficient transcription termination.

PETase is a hydrolase, EC 3.1.1.101, with a polyethylene terephthalate degradation capability. This enzyme was first discovered in the plastic degrading bacterium *Ideonella sakaiensis* as a part of a two-enzyme system, responsible for the hydrolysis of the polymer into a heterogeneous mixture of the respective constituents [5]. Additionally, a PETase codon-optimized sequence for *B. subtilis* , the W159H/F229Y mutant displays an optimal compromise between the catalysis (Kcat) and the Michaelis-Menten (KM) constant, being 9.64 s-1 and 0.08 mmol/L, respectively. The development of the optimized enzyme was made possible by the findings of Xiangxi’s Group [6]. This mutant together with the signal peptide, allows for the excretion of PETase by *B. subtilis* and a fast and efficient PET degradation inside the bioreactor.

MHETase is a hydrolase, EC 3.1.1.102, with a mono-(2-hydroxyethyl) terephthalic acid degradation capability. It is the second constituent of the two-enzyme system used by the plastic degrading bacterium *I. sakaiensis* to degrade PET into its constituents terephthalic acid (TPA) and ethylene glycol (EG). Additionally, a MHEtase sequence codon-optimized for a *B. subtilis* chassis with a W397A mutation displays an optimal compromise between the catalysis (Kcat) and the Michaelis-Menten (Km) constant, being 15.35 s-1 and 1.0 μmol/L, respectively. This mutant was developed by Gottfried’s Group [7].

To further improve our system we aimed to design constructs containing the improved mutants, not just the wildtype enzymes, for both enzymes, however that was not possible during the course of our project. Our constructs are presented in Figure 1 and detailed in Table 1.

![](https://static.igem.org/mediawiki/2021/f/ff/T--NOVA_LxPortugal--wlFIG1_1.jpeg)
![](https://static.igem.org/mediawiki/2021/d/d4/T--NOVA_LxPortugal--wlFIG1_2.jpeg)
**Figure 1:** Constructs maps for both PETase (top) and MHETase (bottom) containing constructs. The PETase construct uses the pBS1C chassis and the MHETase construct uses the pBS2E chassis. Relevant featured sequences are highlighted.

**Table 1:** Details regarding used plasmids; namely features, origin, and iGEM identifier. Constructs are designed for easy exchange of signal peptides and/or genes. Peptide exchange relies on BamHI and ApaI as restriction enzymes whereas gene exchange is dependent on the biobrick exchange. The final step involves connection of the DNA sequences by an appropriate ligase.

![](https://static.igem.org/mediawiki/2021/7/7e/T--NOVA_LxPortugal--wlTABLE1a.jpeg)

The primers used to amplify the insert sequences in the FastCloning protocol have homologous regions with the chassis used that were automatically calculated using the [NEBuilder software](https://nebuilder.neb.com/#!/) and are presented in Table 2. This FastCloning protocol uses the specifically calculated primers in two separate PCR reactions, one for the vector and another for the insert. It is believed that the exonuclease activity of the DNA polymerase produces sticky ends in both PCR products, hence allowing us to fully assemble the construct just by mixing the two PCR products. Finally, this mixture is incubated with a methylation sensitive restriction enzyme to remove the templates and transformed into competent *E. coli* cells.

**Table 2:** Details regarding used primers, namely sequence, annealing temperature (Ta), melting temperature (Tm), and GC content. Ta and Tm were calculated by Tm calculator made available by ThermoFisher Scientific. Upper case letters correspond to the annealing base pairs.

![](https://static.igem.org/mediawiki/2021/8/89/T--NOVA_LxPortugal--wlTABLE2.jpeg)

### *Bacillus subtilis* strains

*Bacillus subtilis* W168 and *Bacillus subtilis* KO7 were used as hosts for gene expression. *B. subtilis* belongs to the genus *Bacillus* and being a member of Gram-positive bacteria, the lack of outer membrane enables the direct secretion of proteins to the growth medium. These proteins are completely transported across the *B. subtilis* membrane as long as they contain an amino-terminal signal peptide that enables them to be secreted. As such this ease of transportation of proteins makes *B. subtilis* of interest in several areas of biotechnology [7].

In this project two strains of *B. subtilis* W168 and *B. subtilis* KO7, were used as hosts for gene expression and an *E. coli* strain was used as an intermediate host. Characteristics of these strains, as well as of the strains produced by the transformation of the latter with our constructs, are presented in Table 3.

The W168 strain, is characterized by being auxotroph only to tryptophan making it one of the most commonly used in academic research and industrial processes due to its ease of transformation providing the basis for *B. subtilis* chassis characterisation [8,9].

The KO7 strain was chosen to be the chassis for the project. This strain’s genotype is characterized by having deletions in 7 protease genes found in WT *B. subtilis* (ΔnprE, ΔaprE, Δepr, Δmpr, ΔnprB, Δvpr and Δbpr) that code for 7 different extracellular proteases responsible for protein degradation [10]. This leads to a higher half-life of the extracellular protein of interest in this project.

**Table 3:** Details regarding the strains utilized throughout our project, namely: host strain, genetic features, antibiotic resistance and optimal growth temperature.

![](https://static.igem.org/mediawiki/2021/2/21/T--NOVA_LxPortugal--wlTABLE3a.jpeg)
![](https://static.igem.org/mediawiki/2021/b/be/T--NOVA_LxPortugal--wlTABLE3b.jpeg)

The single plasmid transformations were performed following the protocol developed by the Uppsala team last year, presented in the Protocols section.

Concomitantly with the results obtained by Uppsala team, hundreds of colonies were obtained in the plates corresponding to the pBS1C transformation, but only a single colony was obtained when using pBS2E (using *B. subtilis* KO7 as host strain). No colonies were obtained when transforming the two plasmids simultaneously. So, the approach suggested but not tested by the Uppsala team was implemented: transforming the cells with pBS2E first and only then with pBS1C. Moreover, small modifications to the Uppsala protocol were employed by culturing the pre-inoculum in LB medium instead of SP medium and excluding the plasmid linearization step. Hundreds of double mutant clones were obtained by implementing this two-step protocol.

### Growth Experiments

A single colony was picked from LB plates and inoculated in 10 mL of liquid LB medium with the appropriate antibiotic(s). The pre-cultures were grown aerobically on a rotary shaker at 37 ºC and 180 rpm, overnight. An appropriate volume of cells was harvested from the pre-culture by centrifugation (10 min at 3000×ɡ), resuspended and then transferred to 250 mL shake flasks with 50 mL of LB medium supplemented with the appropriate antibiotic(s) concentration, yielding an initial optical density at λ600 nm (OD600) of 0.1. These cultures were also cultivated on a rotary shaker at 150 rpm at 28 ºC for 24 h. All cell optical density measurements at 600 nm were performed using the spectrophotometer Ultrospec 10 from Biochrom (Cambridge, UK).

### SDS-PAGE Analysis

After growing for 24 h in shake flasks, a medium sample of each strain was collected and the optical density at λ600 nm (OD600) was measured. For each strain, 1 mL sample with a final OD600 of 0.6 was used for the following steps.

### Intracellular Protein Fraction

Cells were harvested in a microcentrifuge at 5000xg, 10 min. Supernatants were transferred to new tubes to be later used for the extracellular protein extraction protocol, leaving cell pellets intact. Pellets were frozen at -20 ºC for 30 min and then digested with 25 μL digestion buffer, composed of NZY Bacterial Cell Lysis Buffer (NZYTech, Portugal), lysozyme and DNase I (2 µL of lysozyme at 50 mg/mL and 2 µL of DNase I per 1 mL of NZY Bacterial Cell Lysis). Samples were shaken at 200 rpm for 20 min at room temperature. 8.3 μL (in a 1:4 ratio) of Laemmli Sample Buffer (composed of 4x Laemmli buffer and 10 % [v/v] β-Mercaptoethanol) was added to each sample, the resulting mixture was then incubated at 98 ºC for 5 min.

### Extracellular Protein Fraction

10 % (v/v) of trichloroacetic acid (TCA) was added to each supernatant and proteins were allowed to precipitate on ice for 30 min. Samples were centrifuged at 14000 rpm, 5min and supernatant was discarded. Precipitated protein pellets were washed with cold acetone: 200 μL of acetone was added to each tube and tubes were centrifuged again at 14000 rpm, 5 min and acetone was discarded; this step was performed twice. Tubes were incubated at 90 ºC, 5 min, open and inside the hood, to evaporate leftover acetone and dry off pellets. Protein pellet was solubilized in 25 μL of 1X SDS-Glycine Buffer. 8.3 μL (in a 1:4 ratio) Laemmli Sample Buffer (composed of 4x Laemmli buffer and 10% [v/v] β-Mercaptoethanol) was added to each sample, which was then incubated at 98 ºC for 5 min.

### SDS-PAGE Gels

10% SDS-PAGE gels were prepared and 10 μL of each sample and 3 μL of NZYColour Protein Marker II (NZYTech, Portugal) were loaded into the SDS-PAGE gels. Gels were run at 200 V for approximately 1 hour, using 1X SDS-Glycine Buffer. Gels were colored with BlueSafe (NZYTech, Portugal).

PETase and MHETase have a molecular weight of 27.3 kDa and 63.1 kDa, respectively.

### BHET degradation assay

The experiment to evaluate potential of PET hydrolysis by the developed strains was adapted from [11] using the analogue BHET as substrate. The *Bacillus* strains used in this study (Table 3) were grown in LB medium at 28 ºC in shake flasks as described before, after 24 h a medium sample of each strain was centrifuged for 15 min, 4 ºC at 15000 rpm to collect the supernatant to be used as the crude enzyme solution. A reaction mixture of 600 μL was prepared with the following final concentrations: 0.9 mM BHET; 40 mM Na2HPO4·HCl pH7; 80 mM NaCl; 20 % (v/v) DMSO and 2 % (v/v) crude enzyme solution. In a second experiment, the crude enzyme solution was increased to 50 % (v/v). This mixture was incubated at 85 ºC for 2 h, the reaction was stopped by adding 9.6 μL of Na2HPO4·HCl (pH 2.5) and 120 μL of DMSO, followed by an incubation step of 10 min at 85 ºC. The samples were then filtered (2.2-μm filter) to be further analyzed by HPLC.

### HPLC Method

The High Performance Liquid Chromatography (HPLC) method was adapted from [12]. The compounds were quantified using HPLC from Waters (Massachusetts, USA) model ALLIANCE 2695 equipped with an ultraviolet detector model Waters 486 and a symmetry C18 reverse phase column (5 µm 4.6 x 250 mm) of pore size 100 Å. For the analysis, 5 μl of sample was injected using a gradient solution with three solvents: (A) ultra-pure water; (B) Methanol and (C) ultra-pure water with glacial acetic acid 1 % (v/v). The fraction of B decreased linearly from 60 % until 55 % in 10 % of Buffer C from 15 min after injection, returning to the initial conditions for 8 min to equilibrate the column. The flow rate was 1 mL/min. The column temperature was maintained at 25 ºC. BHET and TPA were analyzed at 240 nm.

The calibration curves of TPA and BHET were obtained by injecting standards with known concentrations for each metabolite. Metabolite concentrations in samples were calculated by comparing the peak areas of the samples with the calibration curves.

## Results

### Growth Curve

In Figure 2, the growth-curve for the different strains developed in this work are depicted. These curves were obtained by measuring the optical density of the cultures at different time points.

![](https://static.igem.org/mediawiki/2021/1/1b/T--NOVA_LxPortugal--wlFIG2a.jpeg)
**Figure 2:** Growth-curve profile of the strains developed in this work.

Analysing the growth-curves for the different strains depicted in Figure 2, it is possible to see that the final maximum concentrations achieved in the stationary phase are not very different between each strain. The growth profile of the different strains seem similar, with the exception of BS1 and BS2 strains. Particularly, the lag phase was prolonged when compared with the wild-type KO7. So, it seems that the expression of PETase with both signal peptides had delayed the initial growth. Strangely, this behaviour is not observed when expressing MHETase as well (strain BS6).

### SDS Gel

In Figures 3 and 4 are presented the SDS-PAGE gel results of the different developed strains. Both intracellular and extracellular fractions were analyzed.

![](https://static.igem.org/mediawiki/2021/9/98/T--NOVA_LxPortugal--wlFIG2.jpeg)
**Figure 3:** 10% SDS PAGE gel electrophoresis ran for 1h at 200V colored with BlueSafe. PM - NZYColour Protein Marker II; **A** - *B. subtilis* W168; **B** - *B. subtilis* W168 pBS1C\_SP\_PETase; **C** - *B. subtilis* W168 pBS1C\_AMY\_PETase; Possible bands for PETase highlighted by the yellow box.

![](https://static.igem.org/mediawiki/2021/e/ee/T--NOVA_LxPortugal--wlFIG3.jpeg)
**Figure 4:** 10% SDS PAGE gel electrophoresis ran for 1h at 200V colored with BlueSafe. PM - NZYColour Protein Marker II; **A** - *B. subtilis* KO7; **B** - *B. subtilis* KO7 pBS1C\_SP\_PETase; **C** - *B. subtilis* KO7 pBS1C\_AMY\_PETase; **D** -*B. subtilis* KO7 pBS1C\_MHETase.

After analyzing the Figures 3 and 4, we conclude that in the extracellular fraction of the W168-based strains (wells B and C), a band of the expected size of PETase is visible at 24 h for both signal peptides tested, which is not present in the wild-type strain (well A). Regarding the KO7 cells, no conclusive results were obtained since bands of the expected size are visible in both wild-type and mutant strains for PETase and MHETase.

### Calibration Curves

![](https://static.igem.org/mediawiki/2021/9/9a/T--NOVA_LxPortugal--wlFIG4.jpeg)
**Figure 5:** Graphical representation and table resume of the calibration curves made by HPLC for TPA.

![](https://static.igem.org/mediawiki/2021/a/a2/T--NOVA_LxPortugal--wlFIG5.jpeg)
**Figure 6:** Graphical representation and table resume of the calibration curves made by HPLC for BHET.

The calibration curves obtained had a R2 superior to 0.99 validating the HPLC method implemented to quantify BHET and TPA.

### BHET degradation experiment

**Table 4:** BHET degradation experiment results. Experiment A: 60 μL of crude enzyme was added; Experiment B: 300 μL of crude enzyme was added. n.d.: not detected; BHET: Bis(2-Hydroxyethyl) terephthalate; TPA: Terephthalic acid ; WT: wild-type.

![](https://static.igem.org/mediawiki/2021/d/d3/T--NOVA_LxPortugal--wlTABLE4.jpeg)

Since the initial concentration of BHET supplemented to the reaction mixture was 0.9 mM and the presence of TPA was not detected in all the analyzed samples, we conclude that BHET was not hydrolyzed by the enzymes secreted by the engineered strains in this experiment. Analyzing the results presented in Table 4, we can see that all BHET concentration values are very similar among all strains (including wild-type) and comparable to the initial concentration despite the strain or the final concentration of crude enzyme added to the reaction mixture.

We hypothesize that the amount of enzyme available in the supernatant was not sufficient to degrade BHET and to yield TPA, since bands with the expected protein sizes are barely visible in the SDS-PAGE gels. Even in the experiments where the amount of crude enzyme supernatant was increased to 50 % (v/v) – Experiment B – instead of 2 % (v/v) – Experiment A – no variation in the BHET concentration was obtained. Although it is not referred to in the literature, probably an enzyme concentration step is required in these experiments. We did not find a suitable protocol to apply since all the concentration methods usually imply the enzyme precipitation (like the one described in the preparation of the SDS-PAGE gels) turning unfeasible to further catalytic experiments.

## General Conclusions & Future Perspectives

Although the developed strains failed to degrade BHET, we believe that the optimal conditions to obtain a sufficient amount of enzyme were not achieved due to the lack of time. The conditions used in this work were adapted from the literature where other strains, plasmids and molecular biology designs were implemented. So, the immediate step should be to test various culture conditions and measure the enzyme concentration for several time points to identify the optimal culture conditions for excreting PETase and MHETase by the different strains. We also envision that the engineered cells could be incubated with PET films for several days and further analyze the surface of the films by Scanning electron microscope (SEM) to evaluate the effectiveness of the secreted enzymes in more realistic conditions.

## Protocols

### Competence Development & Transformation in *Bacillus subtilis* (Team UofUppsala 2020)

**Day 0:**

1. From a single colony, inoculate 5 mL of SP medium in a tube and incubate at 37ºC O/N with aeration and 150rpm

**Day 1:**

1. Dilute 1:50 of the O/N cultures on a 16×125 mm test tube (or equivalent) with 500 µl of fresh SP medium
2. Add 1ug of DNA and incubate at 37ºC with aeration, 150 rpm, 5h30min
3. Centrifuge the culture and resuspend in 1 mL of LB medium (pre-heated at 37ºC). Centrifugation + Resuspension is done in an Eppendorf, but the incubation is done in the same tubes as the previous steps.
4. Incubate for 1h30min at 37ºC with aeration and 150 rpm;
5. Plate the whole culture in desired media. Resuspension in 50µl recommended SP.

#### Medium Preparation

**SP Medium (for 10 mL)**

* Spizizen salts 9.215 mL
* Glucose 50% (w/v) 400 μL
* Casein hydrolysate 5% (w/v) 200 μL
* Tryptophan 5mg/ml 100 μL
* Ammonium ferric citrate 22mg/mL 5 μL
* Potassium glutamate 40% (w/v) 50 μL
* MgSO4 1M 30 μL

**Spizizen Salts (for 1 L)**

* K2HPO4 14 g
* KH2PO4 6 g
* Trisodium citrate 1 g
* ddH2O to 1 L

## References

1. Montazer, Z., Habibi Najafi, M. B., & Levin, D. B., Challenges with verifying microbial degradation of polyethylene, Polymers. 12 (2020), 123. https://doi.org/10.3390/polym12010123
2. Jeon, H.J.; Kim, M.N., Functional analysis of alkane hydroxylase system derived from Pseudomonas aeruginosa E7 for low molecular weight polyethylene biodegradation, Int. Biodeterior. Biodegrad., 103 (2015), 141–146. https://doi.org/10.1016/j.ibiod.2015.04.024.
3. Mohanan, N., Montazer, Z., Sharma, P. K., & Levin, D. B., Microbial and enzymatic degradation of synthetic plastics, Frontiers in Microbiology, 11 (2020), 2837. https://doi.org/10.3389/fmicb.2020.580709.
4. C. Li, A. Wen, B. Shen, J. Lu, Y. Huang, and Y. Chang, “FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method.,” BMC Biotechnol., vol. 11, p. 92, Oct. 2011.
5. Maity, W., Maity, S., Bera, S., & Roy, A. (2021). Emerging Roles of PETase and MHETase in the Biodegradation of Plastic Wastes. Applied Biochemistry and Biotechnology, 1-18.
6. Meng, X., Yang, L., Liu, H., Li, Q., Xu, G., Zhang, Y., ... & Tian, J. (2021). Protein engineering of stable IsPETase for PET plastic degradation by Premuse. International Journal of Biological Macromolecules, 180, 667-676.
7. Palm, G. J., Reisky, L., Böttcher, D., Müller, H., Michels, E. A., Walczak, M. C., ... & Weber, G. (2019). Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate. Nature communications, 10(1), 1-10.
8. H. Tjalsma, A. Bolhuis, J.D.H. Jongbloed, S. Bron, J.M. van Dijl, Signal Peptide-Dependent Protein Transport in Bacillus subtilis : a Genome-Based Survey of the Secretome , Microbiol. Mol. Biol. Rev. 64 (2000) 515–547. https://doi.org/10.1128/mmbr.64.3.515-547.2000.
9. J. Heinrich, C. Drewniok, E. Neugebauer, H. Kellner, T. Wiegert, The YoaW signal peptide directs efficient secretion of different heterologous proteins fused to a StrepII-SUMO tag in Bacillus subtilis, Microb. Cell Fact. 18 (2019) 1–15. https://doi.org/10.1186/s12934-019-1078-0.
10. D.R. Zeigler, Z. Prágai, S. Rodriguez, B. Chevreux, A. Muffler, T. Albert, R. Bai, M. Wyss, J.B. Perkins, The origins of 168, W23, and other Bacillus subtilis legacy strains, J. Bacteriol. 190 (2008) 6983–6995. https://doi.org/10.1128/JB.00722-08.
11. X.C. Wu, S.C. Ng, R.I. Near, S.L. Wong, Efficient production of a functional single-chain antidigoxin antibody via an engineered bacillus subtilis expression-secretion system, Bio/Technology. 11 (1993) 71–76. https://doi.org/10.1038/nbt0193-71.
12. D.R. Zeigler, The Bacillus Genetic Stock Center/Bacillus subtilis, Biol. Resour. Model Org. (2019) 35–53. https://doi.org/10.1201/9781315100999-3.

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# Team:NOVA LxPortugal/Results

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