iGEM 2022 — Team upnavarra-spain

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block page\_content %}

During our journey, we have gone through countless days of planification and logistics. Here we can find the tracking of how we developed our project, from start to end.

### July (2021)

19th July - We have our first meeting as a group at midday. We get
presented to iGEM and we also introduce
ourselves to each other. In the afternoon, our instructor Ainhoa and our advisors have a meeting to get the
team started.

21st July - We meet with the PI’s of the past UPNavarra\_Spain team,
2019’s “Color is the new sensor”. We
get introduced to the aim of developing a real application to solve a local problem. In addition, we were
shown iGEM’s parts repository and the different types of human practices. Some key elements of the
competition were explained to us such as the poster, banner, wiki, video presentation, medals and prizes.
Finally, some financing ideas were proposed.

27th July - We decide to search for different local problems which
could be solved by using synthetic
biology as well as past teams’ participations. We considered many approaches but in the end we concluded in
four different ideas: macro farms, vineyard applications, salinity / drought and kits to detect illnesses.
For that, we divided the group into four different subgroups and searched for information.

### August (2021)

13th August - Each of the groups formed presented their advances in
their
quest and later each of the concepts was discussed in regards to their viability. While some ideas such as
salinity / drought continue, macro farms get dropped in favor of creating synthetic meat and vineyard
applications and kits to detect illnesses get merged. Tasks get assigned to continue developing these ideas.
Moreover, due to our future visit to CNTA’s installations we write a small script with the main ideas for
our presentation and the spokeswomen get chosen, Susana and Irene.

27th August - We continue presenting the ideas, this time implementing
information about other teams’ parts, professional investigation groups and businesses. So far, developing a
synthetic steak seems to be leading on. As well, we read the final script for CNTA’s presentation.

### September (2021)

1st September - We finally visit CNTA’s facilities and we introduce
iGEM’s competition. We present our interest in synthetic food to get some feedback from experts, however
their response was not as satisfactory as expected due to the complication of its development. That way, we
decide to discard the idea.

13th September - We take our first photos as an official team.

27-28th September - We take our first photos as an official team.

27-28th September - We attended the autumn course of synthetic biology
in the food sector organized by the chair of synthetic biology to take into account more opportunities for
our project development.

27th September - Our advisors Rosa and Álex go to the Lekaroz
biomeeting with the iGEM EPIC team. This meeting was of vital importance to promote young talent in Navarra,
the bioeconomy and the biorevolution.

### October (2021)

4th October - Mario and María go to MOA Foodtech to make the drawing
for the participation in the 2021 Agar Art contest. Along with them was Hugo, which we did not know was
going to be our future instructor!

13th October - We decided to divide ourselves into groups of three and
kept rotating to brainstorm different ideas for the project. At last, we ended up with different
possibilities and assigned one per couple to search for more information for the following week.

20th October - After many options were laid on the table, Raquel and
Sergio’s option of synthesizing serotonin in a sustainable process by using synthetic biology was chosen!

25th October - We meet to have lunch together and discuss how to
prepare for the trip to go to the 2021 Giant Jamboree in Zaragoza.

### November (2021)

12th November - We go to Zaragoza for the Giant Jamboree where we meet
tons of teams from all over the world and take a chance to see beforehand how the iGEM Jamboree works. On
the way back, we also took advantage and designed our logo and talked about financiation and going to talk
to different businesses of the region.

18th November - Juliana, Irene and Aitor present to the rest of the
group their proposal of presentation to businesses of the project.

21st November - Mario, Sergio and Álex create a massive excel with
decens of businesses and their contacts to see if we could get their financial support.

24th November - We held an online meeting to chat about carrying out a
Christmas raffle to collect money and to search for a lab in our university where we could develop the
project.

28th November - Part of the team travels to Tarragona to the BioNorth
congress where we participated in a full scientific-cultural day and the local scientific talent presented
different papers, talks and a round table.

### December (2021)

1st December - We start developing a survey to spread around the
population to see if the pandemic has a negative effect at a national level.

2nd December - Some team members go to Osasuna’s stadium, our local
soccer team, to talk about the importance of mental health in sport. In addition, we get our tickets printed
and ready to sell them for our Christmas raffle.

20th December - Some, including Susana and Álex, met with Cinfa and we
presented to the company the problem we were facing and the solution we were proposing, they did not
hesitate to help us financially to achieve our final goal.

29th December - A final meeting for the year is held to see how the
sale of tickets is going and to discuss the plans for after the Christmas break.

### January (2022)

6th January - The winner of the raffle was revealed and counted all the
many we gathered.

14th January - Some team members, such as Sergio, and the company AINIA
had a meeting to see how the nanoencapsulated serotonin would affect the microbiota of our organism.

25th January - Due to the international day of the fight against
depression being the 13th of the same month, Mario and Susana made a video explaining the importance of this
illness and the role of serotonin.

31st January - Sergio and Mario go on to create a bank account in
Santander to have all the money from the Christmas raffle together.

### February (2022)

7th February - One of our team members, Irene, got the opportunity to
go to NavarraTV’s programme “La mulga” to talk about the role of women and girls in science on television
with the speakers of the round table we were in the middle of organizing.

8th February - Raquel and Aitor got to go, along with our advisor
Esther and instructor Ainhoa, to the mayoress of Barañain. We had the opportunity to present what iGEM is
about as well as our project.

9th February - The time of the round table due to the day of girls and
women in science finally came. Our iGEM group with the help of NavBiotec (student association of our
University) organized an event called "Women in Biotechnology in Navarra". It consisted of a round table
with the participation of prestigious women scientists from Navarra society.

15th February - We awarded Mikel, our raffle winner with a batch of
gourmet products. As well Adriana got to receive some cheese due to selling the highest amount of tickets.

16th February - We continued discussing how to approach the plasmid
constructions.

17th February - After the biochemical study of serotonin biosynthetic
pathways, we felt the need to better understand the biological role of this hormone in the body. We
therefore met with psychobiologist Laiene Olabarrieta and clinical psychologist José Javier López-Goñi.

23rd February - We had the opportunity to chat with a doctor in
Psychology and university professor, Marta Beranuy, about the worsening of mental health at the population
level as a result of the 2019 pandemic.

28th February - We presented to ANASAPS, a non-profit association
composed of people with mental health problems, family members and all those interested in collaborating
with the promotion of mental health. There we presented our concern for the state of people with mental
health problems, and therefore the reason for the realization of our project.

### March (2022)

1st March - Due to the pillar that the circular economy has in the
project, Javier and Mario, with advisor Rosa and instructor Ainhoa went to the cheese factory Quesos la
Vasco Navarra S.A. The same day our advisor Alex had the opportunity to make visible the importance of
biotechnology in the development of the benefit of society in Rioja TV.

4th March - Member Mario and advisor Álex were interviewed by regional
newspaper Navarra Capital.

5th March - Team leaders Raquel and Sergio met with Joaquín Fernández
Irigoyen, Head of the Navarrabiomed-IdiSNA Proteomics Unit and associate professor at the Public University
of Navarra (UPNA). He provided different methods of proteolysis for the extraction of the essential amino
acid tryptophan, from the proteins that make up the milk whey.

8th March - Thanks to the team’s meeting with professor Eduardo Sánchez
of the Public University of Navarra, we got a closer approach to the terms of circular economy, the
difference between subproducts and residuals; and the cost of opportunity.

14th March - We got the chance to hear from Rosa Martínez and Cristina
Lecumberri, from Verleal, about the residuals from the alimentary industry processing. We specially focused
on the prosecution of broccoli, what parts of the plant are used, when its campaign takes place or
temperature and time of escalation. MOA foodtech’s CEO and associate professor Susana Sánchez took the
chance to give us a quick review about the use of microorganisms to create products of great value with high
amounts of protein using as a source by-products of the food industry.

18th March - The team attended the presentation of the researcher and
head of the Environmental Molecular Microbiology Laboratory of the National Center for Biotechnology,
belonging to the CSIC, Victor de Lorenzo. Afterwards, we had the opportunity to, in private, exchange ideas
on the optimal biobricks to improve our proposed genetic constructs. In addition, we discussed a possible
continuation of the project related to the world of psychobiotics.

23rd March - In the meeting we had with Ingredalia, we were able to
explain what our project consists of and what iGEM is. Due to the proximity of its line of work with the
circular economy part of our project, it was clear that it was a company with which we had to collaborate.

30th March - Pamplona’s city mayor Enrique Maya welcomed members Marta,
Sergio and Raquel, advisor Rosa and instructor Ainhoa to the Pamplona City Hall to talk about the visibility
of mental health and the importance of the circular economy, under the brushstrokes of synthetic biology.
Sergio and Raquel as well tell the group what they discussed with Jokin in their meeting about proteolytic
protocols for tryptophan extraction.

### April (2022)

6th April - A survey was launched and spread to determine the mental
health situation in Spain before and after the COVID19 pandemic.

28th April - Aitor and Juliana were able to be interviewed by the space
that radio COPE Navarra gives to our university to briefly explain what our project consists of and what are
the benefits it brings to our environment.

### May (2022)

5th May - María, Marta and Raquel had the opportunity to visit the
offices of the grouping of municipalities Mancomunidad de Pamplona and chat with the president of the
Mancomunidad.

11th May - We held an online Zoom meeting prior to the final exams to
talk about the last details for the lab work.

30th May - Mario goes to the Agrobiotechnology institute to get the
bacteria strain needed for transformation.

### June (2022)

1st June - Advisors Carlos, Esther and Rosa summon a meeting to explain
that Ainhoa can no longer continue being our instructor.

6th June - Along with other volunteers, members Raquel, Aitor, Susana,
Javier and Mario gave a small workshop to highschool students about the use of molecular techniques to
detect pathogens in crops.

7th June - Members Mikel and Javier assisted to the meeting “Reto
Siberia”, which took place in the CIMA congress hall. Members of Navarra Government such as Ana Ollo,
councilor of citizen relationships and Uxue Itoiz, General Director of Industry, Energy and Strategic
Projects S4 held a speech along with members of the scientific community. The scientific community was
composed of CIMA, CENER and CNTS research centers as well as pioneer business in Navarra
(3P-Biopharmaceuticals, Bread-Free and Telum).

20th June - We met with Estíbaliz and Idoia our primary and secondary
PI’s to get some feedback about the designed plasmid constructions and get some basic knowledge of lab
security.

28th June - We got to the lab to get some audiovisual material and to
start to get more familiar.

30th June - Juliana and Mikel held an online meeting with iGEM’s team
from Waterloo.

### July (2022)

5th July - We met our primary PI Estíbaliz to design the possibility of
evaluating tryptophan’s possible toxicity to bacteria at high concentrations.

19th July - We met again with Estibaliz to redesign the laboratory
approach as time came upon us due to the lack of an instructor due to Ainhoa’s leaving. We as well talk
about the possibility of modeling the chemical reaction of tryptophan’s conversion into serotonin.

24th July - Maria, Aitor and Mikel met lots of teams in the Summer
Online iGEM meeting and gave a small presentation about the project.

30th July - Javier could attend this year’s iGEM Hispanic meetup where
we had the incredible chance to talk about design to use synthetic biology to convert subproducts of the
food industry to serotonin.

### August (2022)

2nd August - We held the first meeting with new instructor Hugo to give
him an update about everything, what the project was about, what we had already done and at which point we
were. The Wiki’s structure started taking shape and the constructions were revised for the IDT order. Search
tasks of different protocols were assigned. As well, many of the deadlines were remembered and plans for
future human practices.

11th August - We revise the state of the Wiki and the data obtained
from the survey launched in April. Primers were designed for following cloning protocols.

10th August - We contacted iGEM TU Delft to translate and fill their
survey.

18th August - We contacted iGEM Thessaly to translate a dictionary full
of synthetic biology related terms.

25th August - The distribution kit finally arrived at the lab.

26th August - We went to our faculty and lab to spend the entire
afternoon recording footage for the teams’ promotional video.

### September (2022)

1st September - We met at the lab to see how we could approach the idea
of using serotonin as a possible medicine and how the idea could be sold.

2nd September - The gblocks finally arrive at the lab.

3rd September - Mikel, Mario and Sergio along with advisor Carlos go to
the presentation of Navarra BG's iGEM project and chat with them afterwards to organize a meeting of both
teams. The same day, we take and upload some pictures that show the different features in the lab to
facilitate access for people with reduced mobility, for iGEM Exeter.

12th September - Javier went to the Agrobiotechnology Institute from
the Higher Scientific Research Center to get the plasmid for the lab work.

17th September - Both UPNavarra\_Spain and NavarraBG finally meet face
to face and exchange their projects and their concerns for the Giant Jamboree, only a month away.

21st September - The primers designed for PCR arrive at the lab.

22nd September - The team gets subdivided into the lab and wiki
subgroups for what's left until freeze day to give a final push.

23rd September - The Wiki group meets at the university’s library to
write down the entirety of the collaborations, human practices and attributions. Tasks get assigned in
regards to contributions, modeling or description among others.

26nd September - A second meeting of the Wiki group takes place after
the weekend to see the positive advances after the weekend in regards to the tasks assigned to each person.

27th September - Maria goes to the university’s official shop to get
free hoodies for the Jamboree. The team made up of Aitor, Aroa and María also has a brief online meeting at
night to separate the integrated human practices and education.

28th September - Primary PI Estibaliz and instructor Hugo go COPE
Navarra's radio station to talk about iGEM and UPNavarra\_Spain’s participation. Another night meeting takes
place between the team members.

29th September - Irene met with Estíbaliz to end the implementation,
and along with Aroa met with Idoia to set a date to start with the project presentation. Aitor wrote a draft
for the future projections and Unai started uploading the attributions and members to the Wiki. Mario wrote
down the different protocols carried out in the laboratory.

30th September - The entire team meets to check the travel plans to go
to Paris, as well as some contributions, judging form and pending things. Aitor, Unai and Javier go to the
European Researcher’s Night to not only explain the technical part and the laboratory work, but also to give
weight to what it means to participate in a competition like iGEM. New CINFA I+D director Isabel Carrilero,
congratulated our team for the great work and presentation performed.

### October (2022)

2nd October - Our parts get added to iGEM’s registry with the optimized
codon usage given by the IDT platform.

3rd October - Maria finishes the design for our official hoodies for
the Jamboree.

6th October - We meet with secondary PI Idoia to get some help and
start organizing the presentation we are going to give in the Jamboree.

8th October - The project description was revised by Hugo and Aitor and
was uploaded to the Wiki. In addition, a front page drawing was designed by Hugo. A quick meeting took place
to check everything that still had to be written down and uploaded to the Wiki.

9th October - After the survey in April, a second survey was launched
and spread to determine the mental health situation in Spain before and after the COVID19 pandemic, and
about circular economy to get a second iteration of the process. The human practices, collaborations and
contributions were also uploaded to the Wiki by Unai.

10th October - The iGEM postcards were sent for iGEM Düsseldorf to
distribute them all over the world.

11th October - The remaining parts of the Wiki are written by everyone
and they are uploaded.

{% endblock %}

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

Along the development of this project, two engineering success cycles have been performed. Related to the
project’s
scope and its general acceptance and market niche, two complete engineering cycles have been completed via
analysis
of
survey data. Regarding wet lab, the team managed to start the first engineering cycle and plan further steps for
the
oncoming ones.

As described above, two surveys were performed. Assessing the validity and relevance of the project was the
purpose
of
the first one. Here, three hypotheses were evaluated. We wanted to check whether mental health has suffered any
deterioration after the COVID-19 pandemic. We also wanted to verify the current awareness regarding mental health,
compared to the one people had five years ago. Last, we aimed to check whether general habits had changed after
lockdowns. Surveys were published and worldwide opinions were gathered and with the input received, we established
that our project was of sufficient relevance and was justified (see Results page). After the data interpretation
of
this first survey, we then launched a second survey with the intention of verifying the awareness of the general
population regarding the use of pharmacological solutions to address mental health diseases and whether people
trust
these solutions. Plus whether people were aware of circular economy solutions, their characteristics and
suitability
for different applications.

This way, we have completed two full cycles of designing, building, testing and learning from our survey results,
that
served the purpose of validating our initial hypothesis and giving our project meaning.

Moving on to laboratory work, the team started the first engineering cycle and managed to design the following
one.

Designing laboratory strategies usually takes longer. Our team took steps very carefully to guarantee design
success.
Design of each construction is described below.

The design of the construct for the synthesis of the Tryptophan Decarboxylase enzyme (TDC) was carried out by
cloning
it into pET28a. pET28a is a non-viral vector of constitutive expression that provides resistance to Kanamycin to
the
bacteria in which it is introduced. It is 5369 bp long and allows the synthesis of proteins with a His Tag at the
N-terminal end. In addition, it has a thrombin cleavage site in case it is desired to eliminate this tail, or to
avoid
leaving additional residues that could modify the synthesized protein.

The cloning strategy to introduce TDC into the vector is based on conventional restriction enzymes (IIR). In this
case, EcoRI and HindIII are used, one at each end. This double digestion ensures that the insert is placed in the
correct direction and not in reverse of the reading pattern.

The insert consists of the human TDC sequence optimized for Escherichia coli, flanked by the corresponding
restriction
sequences, in 5' to EcoRI and in 3' to HindIII, previously added by PCR. In this way, by means of a previous
digestion
of both the insert and the vector, complementary ends are obtained in both, and by means of the T4 ligase, they
are
joined, thus obtaining the construction shown [here](https://benchling.com/s/seq-E9hZnvC5V5NZrifw3ErA/edit?m=slm-k42Tl1plj6GnLHxkSCBP).

The second synthesized enzyme in the project is cloned into a pET15-MHL vector. It is a 7737 base pair long
vector
containing a cloning region between positions 5214 and 7470.
pET15-MHL has ampicillin resistance and the promoter and terminator sequence of phage T7. Moreover, it contains an
additional sequence, corresponding to a 6-histidine tail at the N-terminal end, which helps in the purification of
the
protein of interest.

The cloning strategy performed to obtain the protein of interest is based on the infusion method, whereby by
creating
complementary regions between the ends of the insert and the vector of interest, and treating the mixture with the
corresponding enzymes, a directional recombination is achieved. Insertion of the TPH sequence involves replacement
of
the SacB gene, which provides negative selection of the original plasmid if the treated bacteria are grown in 5%
sucrose-enriched media. Prior to the insertion process, the vector is linearized by the enzyme BseRI whose
restriction
targets are located at positions 5381 and 7370. The final construction sequence can be checked [here](https://benchling.com/s/seq-vZu6iR3AATB5zlnvbGZX/edit).

Comparison of the two cloning methods used:

restriction enzymes (IIR) and ligase-independent cloning, linearizing the plasmid with IIS enzymes.

---

![Responsive image](https://static.igem.wiki/teams/4453/wiki/clonning.png)

---

Constructions results are available in our Results page in our wiki. Unfortunately, our team was not able to test
the
enzyme production capabilities of these constructions, and these assays will be performed in future editions by
upcoming teams who continue with our project. However, we do know that the constructions did not result toxic to
bacterial strains.

For future iterations, we plan to compare the enzyme production capability of our prototype constructions with
ones
designed based on the iGEM Assembly Standards. To do so, we have chosen a set of constitutive promoters, ribosome
binding sites and terminators of different strengths. Following steps involve combining these parts and measuring
the
protein production via qPCR and western blot assays. The chosen parts can be checked in the table below.

---

| Vector | Promoters (different strengths) | RBSs | Terminators |
| --- | --- | --- | --- |
| [BBa\_J04450](http://parts.igem.org/Part%3ABBa_J04450) | [BBa\_J23100](http://parts.igem.org/wiki/index.php/Part%3ABBa_J23100) | [BBa\_B0029](http://parts.igem.org/wiki/index.php/Part%3ABBa_B0029) | [BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015) |
|  | [BBa\_J23102](http://parts.igem.org/wiki/index.php/Part%3ABBa_J23102) |  | [BBa\_B0013](http://parts.igem.org/wiki/index.php?title=Part:BBa_B0013) |
|  | [BBa\_J23105](http://parts.igem.org/wiki/index.php/Part%3ABBa_J23105) |  |  |

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## Our main objective

Our main objective is to get synthetic bacteria containing the two enzymes required for the transformation
of tryptophan extracted from by-products to serotonin:

1. Bacteria with the tryptophan-5-hydroxylase (TPH)
2. Bacteria with the L-amino acid decarboxylase (TDC)

![Responsive image](https://static.igem.wiki/teams/4453/wiki/experiments/exp-img1.png)

---

When these two types of synthetic bacteria express their enzymes of interest and these ones are put
together in a test tube with the necessary amount of by-products-coming tryptophan, the TPH enzyme is able
to convert it into the intermediate 5-hydroxytryptophan and the TDC enzyme gets it to transform it into
5-hydroxytryptamine, better known as serotonin.

The lab procedure will be divided into two cloning secondary experiments:

### 1. Synthetic bacteria expressing TDC

The obtaining of this bacterium is achieved by means of the following procedures, based on restriction
enzymes and ligase cloning:

![Responsive image](https://static.igem.wiki/teams/4453/wiki/experiments/exp-img2.png)

Miniprep of the pET28a plasmid

First of all, it is needed to extract the DNA plasmid corresponding to the pET28a vector from bacteria
containing it. It is also required to ensure the correct extraction by gel electrophoresis. It corresponds
to the 5369 bp band.

Digestion and purification of pET28a

Once the vector is obtained, it must be digested with the corresponding restriction enzymes: EcoRI and
HindIII, which will leave sticky ends at both sites of the linearized vector. Then, a gel purification is
required to ensure the digestion of the vector.

![Responsive image](https://static.igem.wiki/teams/4453/wiki/experiments/exp-img3.png)

Zoom of the region where the restriction enzymes cut the plasmid:

![Responsive image](https://static.igem.wiki/teams/4453/wiki/experiments/exp-img4.png)

PCR of TDC

As the gBlock was ordered just with the coding sequence, it is required to add the restriction sites of
EcoRI and HindIII at the ends flanking it. Concretely, EcoRI site must be at the 5’ end and HindIII at the
3’ end.

Digestion and purification of TDC

The next step is the digestion of the TDC fragment flanked by the restriction sites with the corresponding
enzymes. Therefore, we will have the TDC gene with the necessary sticky endings to perform the ligation. It
must also be purified after the diagnostic gel.

Ligation of TDC and pET28a

Then, the digested and purified TDC and pET28a must be ligated with the T4 ligase.

Transformation of bacteria and plating

The transformation must be performed with the ligation containing the gene of interest. The bacteria
selected are E.coli and the methods of transformation are heat shock and electroporation, in order to
increase our probabilities of success by developing different ways of transformation. Then, the synthetic
bacteria are platted in agar with kanamycin, as the plasmid contains the gene for kanamycin resistance.
Therefore, a positive selection will take place because only the transformed bacteria that contain the
plasmid will be able to survive and grow in that medium.

Purification of the enzyme

The next step is to purify the enzyme expressed by the bacteria thanks to the histidine tag it owns that
will bind to the metal ions immobilized in the resin matrix of purification columns. This is called His-Tag
purification, which is based on the affinity of histidines residues for metal ions (added at the C-terminal
of the protein because of its previous cloning in pET28a).

### 2. Synthetic bacteria expressing TPH

The obtaining of this bacterium is achieved by means of LIC cloning.
The following procedures are required:

LIC or ligase-independent cloning is a strategy which relies on the 3'-5' exonuclease activity of T4
polymerase. Although the polymerase activity of T4 prevails over its exonuclease activity, this predominance
can be reversed by modifying the reaction conditions. For this, it is necessary that the ends of the vector
and the insert of interest are complementary and are composed of all the dNTPs except for one of them, which
should appear once at the end of these borders. By PCR, we can add to the insert of interest the sequence
complementary to the flanking regions around the insertion site in the vector.

If the reaction mix contains all the required elements with only the dNTP complementary to the one that
appears uniquely at these borders, the polymerase will use its 3' → 5' exonuclease activity to digest the
strand it is working on to the point where it can add the nucleotide from the mix. At that point where it
can add a nucleotide because of the presence of a template, the polymerase activity will precede over the
exonuclease and incorporate the nucleotide that has been added to the reaction. In this way, large overhangs
are generated that will allow binding between the vector and the insert of interest without the need for a
complementarity ligase enzyme. For this, these overhangs must be at least 15 nt in length.

Let’s say that the nucleotide of choice is thymine, T. With the sequences flanking the insertion site,
primers are designed with which to add this complementarity to both sides of the insert of interest. These
sequences are composed of all possible nucleotides and have a single T at the end. At the start of the
reaction, the T4 enzyme will begin to digest one of the strands due to the lack of nucleotides in the mix.
This enzyme will not recover its polymerase activity until it reaches the T position, where it can insert
its complementary base, A, which is the only one containing the reaction cocktail (dATP). This will create
single-stranded ends of approximately 15 nt in length in both the insert and the vector that will be
complementary to each other and will bind immediately, without the need for ligase.

A variant of the LIC method is the InFusion method, which consists of a commercial kit with an enzyme that
has this intrinsic capacity, so that with a commercial mix and correctly designed primers, it acts as a
polymerase/exonuclease, generating these cohesive ends necessary to generate the complementarity in both the
insert and the vector, and to get them to join without the need for subsequent ligation.

![Responsive image](https://static.igem.wiki/teams/4453/wiki/experiments/exp-img5.png)

The obtaining of this bacterium is achieved by means of the following procedures:

Miniprep of the pET15-MHL plasmid

The first step is based on the isolation of the pET15-MHL plasmid from the bacteria.

![Responsive image](https://static.igem.wiki/teams/4453/wiki/experiments/exp-img6.png)

Linearization and purification of pET15-MHL

The plasmid pET15-MHL must be linearized by digestion using BseRI (type II restriction enzyme) and then
purified by gel electrophoresis.

![Responsive image](https://static.igem.wiki/teams/4453/wiki/experiments/exp-img7.png)

PCR of TPH

On the other hand, the insert requires the addition of overlapping ends in order to be able to join the
plasmid ends resulting from linearization. These ends consist of complementary 15 nucleotide DNA sequences.

T4 polymerase reaction using 3’ exonuclease activity

First, a reaction must take place between T4 polymerase and the linearized pET15-MHL, in the presence of
dGTPs. The same reaction must take place with TPH but in the presence of dCTPs, instead of dGTPs.

Annealing between TPH and pET15-MHL

Then, both parts join together as they present 15 bp overlapping ends. No ligase is required for this step,
and this is the reason for the cloning method’s name.

Transformation of bacteria and plating

The resulting construction must be introduced inside the competent bacteria (TOP10) by heat shock. Then,
the synthetic bacteria are platted in agar with ampicillin, as the plasmid contains the corresponding
resistance gene. Therefore, a positive selection will take place because only the transformed bacteria that
contain the plasmid will be able to survive and grow in that medium.

Purification of the enzyme

The next step is to purify the enzyme expressed by the bacteria thanks to the histidine tag it owns that
will bind to the metal ions immobilized in the resin matrix of purification columns. The histidine tag is
added at the N-terminal of the protein because of its previous cloning in pET15-MHL.

### 3. Serotonin production

Once we have the two purified enzymes: TPH and TDC, we have the need to obtain our raw material, which is
the tryptophan. This must be extracted from by-products, such as broccoli or milk whey.

Then, the in vitro serotonin production will take place. This reaction requires both enzymes and the
tryptophan as precursor. The aromatic amino acid will be hydroxylated to 5-hydroxytryptophan (5-HTP) by TPH.
The 5-HTP acts as an intermediate as it can be transformed by TDC to 5-hydroxytryptamine, more commonly
known as serotonin. Eventually, the serotonin is extracted by using chromatographic techniques,
specifically, high performance liquid chromatography (HPLC).

![Responsive image](https://static.igem.wiki/teams/4453/wiki/experiments/exp-img8.png)

At the end, this serotonin can be nanoencapsulated in non-polluting biological enclosures with the aid of local experts in nanotechnology and biotechnology. This way, our nanocapsules of serotonin could be used for helping in the recovery of mental disorders patients as a potential supplement, thus contributing to the improvement of global mental health and local economy in an eco-friendly way.

And this is the biological basis of our circular economy solution to fight depression!

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{% block title %}Results{% endblock %}
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This being our project’s first year, our team has focused on setting a solid foundation from which future teams
will be able to continue with it. This purposefully is an initiative that we have planned to finalise in subsequent
iGEM editions, as it necessarily needs to be concluded in a larger time span.

Our team has started this journey by ensuring the validity of our project and setting some key starting points for
future wet lab steps. The validity of our project has been hypothesised by our team members, and the market niche
for our potential product has been analysed by our data scientist by market researching. Both of these have been
proven via survey launch and analysis of the data retrieved from them.

### Project justification

As our project was focused on a dietary supplement to treat anxiety and depression we wanted to see the
current situation of these mental illnesses. Thus, a survey was made to examine Spain’s mental health. Not
only were we keen on knowing its current situation, but also, its evolution before and after the pandemic,
and, mainly, the lockdown.

We defined three main hipotesis to corroborate our suspicions:

1. Hypothesis 1: Mental Health has deteriorated since the COVID-19
   pandemic.
2. Hypothesis 2: Society is nowadays more aware of mental health than 5 years
   ago.
3. Hypothesis 3: Society’s habits and dynamics have changed due to the
   COVID-19 pandemic

Regarding the first hypothesis, we found in questions five and six, related to the first hypothesis, how
the percentages of agreement and disagreement reflect the worsening of the respondents' mental health.

![Responsive image](https://static.igem.wiki/teams/4453/wiki/results/image6.png)

Also, we studied with a factorial analysis both hypothesis one and three as a whole. This analysis
suggested that questions 13 and 14 had great importance in an individual’s mental health based on the
factors and characteristics of the model. Notice that the importance of the question is determined by the
length of the arrow in the plot below.

![Responsive image](https://static.igem.wiki/teams/4453/wiki/results/image2-2.png)

Factors from one to six, related to nutrition, sleep, sports habits and so on, had a differential weight
when compared with habits related to addiction and narcotics. We suspect an error related to social
convenience here, as two last topics are a touchy subject for most people.

The following figure shows how responses related to good habits decrease in some of the factors discussed
(in green), thus increasing the number of responses related to bad habits (in red and orange) with the
passage of the pandemic.

![Responsive image](https://static.igem.wiki/teams/4453/wiki/results/image3.png)

However, contrary to what we suspected, as the figure below suggests, respondents are not used to talking
about their mental health with whom, at first, would seem to be their closest people. This response,
however, only reinforces our initial thought when we launched our project: Mental health is still a taboo
topic and it needs to be acknowledged as any other disease for it to be treated with more ease.

![Responsive image](https://static.igem.wiki/teams/4453/wiki/results/image7.png)

We defined a second survey to delve deeper into aspects of interest that we did not address in the first one, such as:

1. Hypothesis 1: Whether the tools provided and current education around mental health issues are accurate.
2. Hypothesis 2: Whether a respondent would be willing to take a supplement or drug to treat a clinical depression or similar condition.
3. Hypothesis 3: Whether a respondent would be willing to take a supplement or product from the circular economy.

Further data retrieved from our second survey however showed that this would be this way due to the lack of
information and resources people have to treat these disorders (figure below).

![Responsive image](https://static.igem.wiki/teams/4453/wiki/results/image5.png)

Although they do not feel they have adequate resources, most respondents are willing to treat their disease
with therapeutics.

![Responsive image](https://static.igem.wiki/teams/4453/wiki/results/form21.png)

Similarly, the majority of respondents believe that these treatments are beneficial to their health, with
only 18% of respondents being indifferent.

![Responsive image](https://static.igem.wiki/teams/4453/wiki/results/form22.png)

Regarding circular economy initiatives, we have deduced that, in general, people are in fact positive about
circular economy and its potential applications (see figure below). So we are one step further to confirm
that our product has, indeed, a place. Further questions in our survey dove deeper into how familiar society
is to circular economy strategies and the vision they have on them.

![Responsive image](https://static.igem.wiki/teams/4453/wiki/results/image9.png)

In fact, our data suggests that there is a believe in our society about circular economy solutions being
beneficial (see figure below). This result was partially expected, as these have been widely advertised and
have been of great impact lately. However, we did want to be sure about the potential impact our product
would have once launched, when it comes to its awareness and social rejection.

![Responsive image](https://static.igem.wiki/teams/4453/wiki/results/image1.png)

Moreover, we have seen a general willingness to try solutions that have emerged from circular economy
solutions (figure below).

![Responsive image](https://static.igem.wiki/teams/4453/wiki/results/image4.png)

Nevertheless, we do see a decrease when it comes to the willingness to take a medicine or supplement
derived from circular economy strategies (Figure below). However, we lack data indicating whether this
behavior originates from the general apprehension against medicines or from a distrust of circular economy
solutions in these areas in particular. But, noticing the tendencies from previous questions, we could infer
that it is more probable to be as it is due to a general uneasiness to medicines (data not shown).

![Responsive image](https://static.igem.wiki/teams/4453/wiki/results/image8.png)

### Model

#### Introduction

After analyzing our first survey we were keen on extracting more abstract knowledge. This is why we
developed a model to further learn characteristics of our data. Due to the nature of the survey, the most
appropriate category of learning was clustering. To perform this technique, we needed to study the
correlation of the data, and process the data, for the training phase of the algorithm. We use the
topological algorithm DBSCAN to find groups in the data after applying principal component analysis.

#### Choosing principal components

First, we select the principal components on which we are going to apply the clustering algorithm. It can
be seen that when projecting the data on the first component and any of the other two we do not have a clear
division of the data.

![Responsive image](https://static.igem.wiki/teams/4453/wiki/model/scatterxy.png)

![Responsive image](https://static.igem.wiki/teams/4453/wiki/model/scatterxz.png)

Although, when projecting on the second and third component there seems to be a division into two groups

![Responsive image](https://static.igem.wiki/teams/4453/wiki/model/scatteryz.png)

Analyzing the mean of this variable we see that in cluster 0 there is a tendency to have less discomfort in talking about mental health problems as opposed to cluster 1. In addition, in job/classmate in cluster 0 there is a tendency not to have discomfort with work/classmates while in cluster 1 there is more reticence, this is related to what was commented with the previous variable.
In the rest of the variables the differences are not very significant, so they do not represent an element to differentiate the individuals.
Individuals marked as -1 do not belong to any cluster.

### Wet lab

The main objective this year was to provide a firm starting point for the next steps in the project to be
taken with more ease. Thus, our team has designed functional base DNA constructions with the two main
enzymes that participate in serotonin biosynthesis in humans: Tryptophan hydroxylase (TPH) and tryptophan
decarboxylase (TDC).

The cloning strategy to introduce TDC into the pET28a vector was based on conventional restriction enzymes
(IIR). In this case, EcoRI and HindIII were used, one at each end. This double digestion ensures that the
insert was placed in the correct direction and not in reverse of the reading pattern.

The insert consists of the human TDC sequence optimised for Escherichia coli, flanked by the corresponding
restriction sequences, in 5' to EcoRI and in 3' to HindIII, previously added by PCR. In this way, by means
of a previous digestion of both the insert and the vector, complementary ends are obtained in both, and by
means of the T4 ligase, they are joined.

The cloning strategy performed to obtain the TPH protein of interest is based on the infusion method,
whereby by creating complementary regions between the ends of the insert and the pET15-MHL, and treating the
mixture with the corresponding enzymes, a directional recombination is achieved. Insertion of the TPH
sequence involves replacement of the SacB gene, which provides negative selection of the original plasmid if
the treated bacteria are grown in 5% sucrose-enriched media. Prior to the insertion process, the vector is
linearized by the enzyme BseRI whose restriction targets are located at positions 5381 and 7370.
The employed sequences are available in Table 1.

Cloning of the pET28a-TDC construction was done by means of standard molecular biology techniques (see
Engineering page in our wiki). Briefly, pET28a vector was extracted from the strain containing it. Vector and
insert were processed to perform the ligation reaction, and the final construction was electroporated into E.
coli TOP cells for long term storage.

Construction of the pET15-MHL-TPH construct did not result possible. This was mainly, as we suppose, due to
our team not being familiar to the technique. In synthetic biology issues, this could mean a poor
understanding when it comes to the DNA fragment design, problems regarding experimental conditions and
repetitivity, or just the need of more time to assimilate its steps. Sadly, our team lacked the time to
diagnose what was going wrong with their technique, so in the end we had to abort the experiment.

---

Table 1: Sequences used in wet lab

| TPH optimised for its production in E. coli | URL or sequence (5’ to 3’) |
| --- | --- |
| TPH optimised for its production in E. coli [Link](https://benchling.com/s/seq-6Q7xLQAGonURGa0qtJw5/edit) | |
| TDC optimised for its production in E. coli [Link](https://benchling.com/s/seq-vj2D0qLdGVeqqMAQ6brf/edit) | |
| Primer FwTDC ATCCGAATTCATGGACATTGAG | |
| Primer RvTDC CCGCAAGCTTTTATTGAACGTC | |
| Primer FwTPH TTGTATTTCCAGGGCATGTGTGAAA | |
| Primer RvTPH CAAGCTTCGTCATCACTAGATCGACTGGTC | |

---

Note that the constructions here obtained do not meet the Biobrick Assembly Standards. Our lab personnel
were unfamiliar with these strategies and, as a starting point, it was established that it would be better
to store the constructions in common commercial vectors, more familiar to the ones used in the everyday
procedures in our lab. The parts submitted to the iGEM Part Registry, however, have been done so that there
are no modifications with regards to the coding sequences we have employed, but require the Assembly
Standards characteristics. In fact, the main objective for future editions will be to compare different
constructions that do meet the Standards to optimise enzyme production in relation to the more common
vectors employed here (see Proposed Implementation page in our wiki).

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