iGEM 2022 — Team tec-chihuahua

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{% block title %}Notebook{% endblock %}
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**# **Notebook****

---

Here is an overview over the accomplishments and the challenges we had to manage during our project. Click on each month to see how our project developed week by week.

November
December
January
February
March
April
May
June
July
August
September
October

![]()

November

Week 22nd - 28th

December

Week 13rd - 19th

January

Week 17th - 23rd
Week 24th - 30th

February

Week 31st - 6th
Week 7th - 13rd
Week 14th - 20th
Week 21st - 27th

March

Week 28th - 6th
Week 7th - 13rd
Week 14th - 20th
Week 21st - 27th
Week 28th - 3rd

April

Week 4th - 10th
Week 11st - 17th
Week 18th - 24th
Week 25th - 1st

May

Week 2nd - 8th
Week 9th - 15th
Week 16th - 22th
Week 23th - 29th

June

Week 30th - 5th
Week 6th - 12th
Week 13th - 19th
Week 20th - 26th
Week 27th - 3rd

July

Week 4th - 10th
Week 11th - 17th
Week 18th - 24th
Week 25th - 31th

August

Week 1st - 7th
Week 8th - 14th
Week 15th - 21th
Week 22th - 28th

September

Week 29th - 4th
Week 5th - 11th
Week 12th - 18th
Week 19th - 25th
Week 26th - 2nd

October

Week 3rd - 9th
Week 10th - 12th

![Main crops produced in Mexico: Chilli, Corn, Beans and Onions.](https://static.igem.wiki/teams/4147/wiki/notebook.png)

## Week of December 21th to 27th

### Project

This week corresponded to the first week of the end-of-year vacations. We continued to
develop our application projects while trying to find other potential topics.

## Week of December 28th to 03rd

### Project

During the last week of the year we continued our research work on topics.

**## **Week of November 22nd to 28th****

Our iGEM team is formed! After a selection process, the team member list was published.

![Our University's, Tec de Monterrey, list for the iGEM 2022 team.](https://static.igem.wiki/teams/4147/wiki/notebook/nov22-28.png)

**## **Week of December 13th to 19th****

We had our first meeting, but it was a short one in which we talked a bit about the formation of the new team. We took the rest of the month as vacations.

**## **Week of January 17th to 23th****

18th: We had a more formal first meeting. We discussed some administrative things since iGEM is a student group at our university.

19th: We planned out fundraising activities to do during the semester.

**## **Week of January 24th to 30th****

27: We made an inventory and a preliminary shopping list with things we might need for the project.

**## **Week of January 31st to February 6th****

4th: It had been 2 years since our campus participated in iGEM. Thus, we looked out for previous projects from 2021 to catch up on the competition.

Also, Alessandra joined the team!

**## **Week of February 7th to 13th****

We did some fundraising activities, such as a raffle for Valentine’s Day.

**## **Week of February 14th to 20th****

16th: iGEM at Global Women´s Breakfast! On our campus, Marifer and Elizabeth attended the event and volunteered to help the organizing committee.

18th: We previously formed smaller teams and started brainstorming ideas for our project. We had a reunion with our advisor and PI so they could give us feedback. Our first ideas consisted on:

* A diagnostic mechanism for detecting polycystic ovarian syndrome.
* A biofungicide against Texas root rot.

  * A novel procedure for producing snake antivenom.
  * A biofungicide against chilli wilt.

![Logos that define each of the ideas. Also, a zoom meetig with our advisor and PI.](https://static.igem.wiki/teams/4147/wiki/notebook/feb14-20.png)

19th: We started training in the entrepreneurship area. We attended our first MasterClass about value proposals.

![Masterclass poster: Value proposal - The heart of business models.](https://static.igem.wiki/teams/4147/wiki/notebook/feb14-20-1.png)

**## **Week of February 21th to 27th****

23rd: We had our first meeting in person! It worked as a team-building experience.

![Picture of all the team members at the moment and PI. First meeting in person.](https://static.igem.wiki/teams/4147/wiki/notebook/feb21-27.png)

26th: We continued our entrepreneurship training. This day´s Master Class was about creating a business in a digital ecosystem.

![Masterclass poster: Creating a business in a digital ecosystem.](https://static.igem.wiki/teams/4147/wiki/notebook/feb21-27-1.png)

**## **Week of February 28th to March 6th****

2nd: We had a follow up reunion to present our ideas for the project. We received feedback from advisors and our PI once again.

5th: We continued our entrepreneurship training with a Master Class about capital raising.

![Masterclass poster: Capital raising](https://static.igem.wiki/teams/4147/wiki/notebook/march28-6.png)

**## **Week of March 7th to 13th****

9th: We continue exposing our project ideas. Once again, we received feedback. Also, many of our women teammates took part in a national movement called “El nueve ninguna se mueve”. During this day, women cease their daily activities as a way of protesting against the high femicides that occur in Mexico.

**## **Week of March 14th to 20th****

16th: We continued with our project idea brainstorm. We presented advances to our advisors and instructor.

![Presentation of ideas in front of advisors and PI.](https://static.igem.wiki/teams/4147/wiki/notebook/march14-20.png)

**## **Week of March 21th to 27th****

22nd: We gave a class to high school students about synthetic biology and introduced them to iGEM.

![Class made for high school studets at our campus.](https://static.igem.wiki/teams/4147/wiki/notebook/march21-27.png)

23rd: Our team is made up of several members with no previous experience in genetic engineering. Thus, we made a short presentation to introduce them to important concepts. We also had our final presentation of project proposals. We presented them to our advisors, instructors, and our PI´s special judges.

![Final formal presentation in front of advisors and PI.](https://static.igem.wiki/teams/4147/wiki/notebook/march21-27-1.png)

It was also our PI´s birthday!

![PI and team member with a birthday pie.](https://static.igem.wiki/teams/4147/wiki/notebook/march21-27-2.png)

24th: We continued with our classes for high school students. This time, we made a workshop in which we talked about synthetic biology and expression vectors. We carried out an activity in which the students had to create expression systems for different situations.

27th: We sold hamburgers as a fundraising event. We made over 360 burgers in a single day and it was a team-building experience.

![Two photos of the team at a team member's house to make hamburguers.](https://static.igem.wiki/teams/4147/wiki/notebook/march21-27-3.png)

**## **Week of March 28th to April 3rd****

31st: iGEM at Women LeadING STEM possibilities! This was an event to make visible the participation of women in engineering and inspire future generations. Our team-mates shared their most significant achievements and experiences in their lives. Cesar and Alejandro just took pictures

![Foto on the left is the poster for the event. Foto on the right is the team members in the auditorium of the event.](https://static.igem.wiki/teams/4147/wiki/notebook/april28-3.png)

April 1st: We officially have a project! Although each idea was very unique and interesting, it was decided to focus on Agrocapsi: A biofungicide against Phytophthora capsici.

It was also Cesar´s birthday!

![All the team members and Cesar with a birthday pie.](https://static.igem.wiki/teams/4147/wiki/notebook/april28-3-2.png)

**## **Week of April 4th to 10th****

This week we didn't have a reunion with advisors or our PI, so we just continued doing research about our project.

**## **Week of April 11th to 17th****

We didn’t have a reunion this week, but continued with our research.

**## **Week of April 18th to 24th****

22nd & 23rd: We presented our project in Biohack. This is a bioentrepreneurship competition held at our institution. We earned 3rd place!

It was also Alejandro´s birthday

![Some team members at the Biohack event, after receiving the third place.](https://static.igem.wiki/teams/4147/wiki/notebook/april18-24.png)

**## **Week of April 25th to May 1st****

28th: Tec-Chihuahua was officially in iGEM!

![E-mail received by iGEM of confirmation that the team is officialy signed-up.](https://static.igem.wiki/teams/4147/wiki/notebook/april25-1.png)

29th: During this meeting we planned some more fundraising events activities.

**## **Week of May 2nd to 8th****

6th: We presented our project for the last time to our advisors and instructors. This time, we also shared our plans for human practices, project modeling, entrepreneurship, and all areas of an iGEM project.

![First time presenting the proyect after selecting it from the multiple ideas we brainstormed. It was a formal presentation in from of advisors.](https://static.igem.wiki/teams/4147/wiki/notebook/may2-8.png)

**## **Week of May 9th to 15th****

13th: We established our team areas! Our team was divided into Human Practices & Entrepreneurship, Wet Lab, Model and Wiki. We voted for area leaders and for our team leader.

**## **Week of May 16th to 22th****

18th: We took a brief programming class from Denisse so we could start working on the wiki.
Judith fell asleep.

![Team members in a room paying attention to Denisse's instructions.](https://static.igem.wiki/teams/4147/wiki/notebook/may16-22.png)

19th: Lab capacitation started! Cesar and Daniela helped us practice some essential things at the lab. We also had a reunion with new instructors (Alan, Ana Laura, and Ana Victoria). Thus, we made a brief introduction about our project and Human Practices plans.

![Lab team members reunited in a lab, while Cesar is teaching basic lab concepts.](https://static.igem.wiki/teams/4147/wiki/notebook/may16-22-1.png)

21st: Our team was present at Solved! This was an event to show attendants our work at the engineering school. We talked to many people about the application of synthetic biology and about iGEM.

![Team members in front of our poster we presented at SOLVED.](https://static.igem.wiki/teams/4147/wiki/notebook/may16-22-2.png)

20th, 21st & 22nd: We participated in an educational hackathon. It was an opportunity to meet with teachers and experts in pedagogy. We also learned about the main problems of education in our country.

![Three fotos of different team members at different moments during the event. In the middle foto we can see Chuy give a speech in front of the audience.](https://static.igem.wiki/teams/4147/wiki/notebook/may16-22-3.png)

**## **Week of May 23th to 29th****

During this week the model team established goals and objectives for this year´s project. We decided to work on a system of ODEs to describe the gene expression of our system. We also designed some future ideas if there was time available.

24th, 25th & 26th: We participated in Innovation Week. We learned subjects related to entrepreneurship, innovation, and technology. We also made contacts with entrepreneurs from various areas.

![Innovation Week's poster.](https://static.igem.wiki/teams/4147/wiki/notebook/may23-29.png)

27th: We continued our wiki capacitation with Denisse.

Also, iRNAldo joined our team!

![iRNAldo.](https://static.igem.wiki/teams/4147/wiki/irnaldo.png)

**## **Week of May 30th to June 5th****

2nd: PhD Carlos Hirales joined our team as a secondary PI. Thus, we had a meeting with him where we presented our project and the wet lab methodology.

**## **Week of June 6th to 12th****

This week the Human Practices team collected contacts to form a directory and made a promotional pitch. The modeling team also made a preliminary diagram of how our genetic circuit works.

![Whiteboard with innitial genetic circuit diagram.](https://static.igem.wiki/teams/4147/wiki/notebook/june6-12.png)

8th: We participated in Expo Ingenierías, where we had a space to talk about what is iGEM and our team´s project to people at our campus.

![Two team members in front of a screen and a poster of our campus trayectory of iGEM team members through the years.](https://static.igem.wiki/teams/4147/wiki/notebook/june6-12-1.png)

**## **Week of June 13th to 19th****

The Human Practices team redacted a draft for the interviews to producers and started contacting them. On modeling, we started working on our system of ODEs and also had a meeting with UAM.

![Zoom meeting with a couple of team members from UAM's team.](https://static.igem.wiki/teams/4147/wiki/notebook/june13-19.png)

**## **Week of June 20th to 26th****

During this week the Math model team finished their system of ODEs!

![Denisse in front of a witheboard with differential equations.](https://static.igem.wiki/teams/4147/wiki/notebook/june20-26.png)

23rd: We performed several interviews in different cities for our Human Practices. We also attented a chilli demonstration! we had the opportunity to talk with producers and agriculture-focused companies.

![Picture on the left are some team members in a field in their way to a meeting. On the left is a poster of said meeting.](https://static.igem.wiki/teams/4147/wiki/notebook/june20-26-1.png)

**## **Week of June 27th to July 3rd****

28th: Saghir joined the team!

30th: Tec-Chihuahua was at Beautiful Patterns! We had the amazing opportunity of talking to a group of girls from all over the country. We made a brief introduction for them to synthetic biology and iGEM.

![Participation of three members in the zoom event of Beautiful Patterns.](https://static.igem.wiki/teams/4147/wiki/notebook/june27-3.png)

**## **Week of July 4th to 10th****

The Human Practices started working on a manual for chilli producers. They also met with Tec Guadalajara and a team for California to make collaborations. Several interviews were also made. The Wet Lab team prepared some culture media, antibiotics and practiced how to run an SDS-PAGE.

The Model team had a meeting with the TecMonterrey\_GDL team. In this meeting we brainstormed about what they could model, and we also helped them to find examples that they could use as a base.

The entrepreneurship area works with the Physical laws. To understand the operation of our product it is necessary to go back to the physical laws that intervene in biopesticide, it is for this reason that we carry out a bibliographic analysis and apply a little analysis and internal reflection in our knowledge to be able to determine the probable mechanism of action of a biopesticide.

**## **Week of July 11th to 17th****

The Human Practices team finished working on a synthetic biology manual. They also had several meetings with teams such as UAM, Tec CEM and HKU.

The Wet Lab team prepared chemically competent cells and transformed the following parts:

* From 2021: 16M (plate 5), 4E (plate 3), 3G (plate 3), 1J (plate 2), 4K (plate 3), 17D (plate 1)
* From 2019: 5G (plate 6), 1J (plate 2), 1I (plate 6), 17D (plate 1), 16M (plate 5), 3G (plate 3), 4K (plate 3)
* From 2018: 2J (plate 2), 17D (plate 1), 15N (plate 5), 6D (plate 2). 4G (plate 3), 3L (plate 3)

The Model team solved the differential equations using MATLAB. We also carried out our first engineering cycle in the area!

The entrepreneurship area works Problem One of the most important steps or processes is to know as much as possible about the problem. Ask ourselves what?, how?, when?, where? and why? It will help us determine possible solutions to this problem. That is why it was carried out in a bibliographic way and the field visits were used to know in depth the wilt caused by Phytophthora capsici. This compendium of information focused on the problem section of the project.

**## **Week of July 18th to 24th****

The Wet Lab team made and transformed the vector pUC19 with part 16M and a lacI regulated promoter which we characterized.

The Model team alongside team UAM started working on a manual on how to start a mathematical model. The main idea was to share what we had learned to help future teams.

The entrepreneurship area works Preliminary hypothesis After having known the problem, it is easier to brainstorm possible solutions for the resolution of this problem. That is why in addition to our proposal, other possible solution alternatives were created such as genetically modified chili plants that grant resistance to the pathogen or genetically modified biological control that produced antimicrobial peptides against the pathogen.

**## **Week of July 25th to 31th****

26th: We sent to iGEM - Tec CEM samples of water taken from the water dam Reliz located in Chihuahua. We sent 2100 mL of these samples.

The Wet Lab team modified the previous assembly strategy. This time they followed a standard assembly and transformed again on the same strain. Also started an IPTG induction of the grown colonies.

In addition to our system of differential equations, the Model team began building the mathematical model in Simbiology.

28th: We reached ICHEA (Instituto Chihuahuense de Educación para Adultos), which is an institution dedicated to education for adults, to see if they could help us out with the translation of our comic and synthetic biology manual to rarámuri. Rarámuri is a native language for an ethnic group here in Chihuahua. They were happy to help and they even offered to translate it to ODAMI as well, another ethnic language.

30 & 31st: We went out to the streets asking for financial support. People really supported our cause!
The entrepreneurship area works with the Product Development deliverable. Product development is a fundamental part of sustainable development as it aims to change the linear economic model to a circular one to reduce the exploitation of natural resources and environmental pollution. This is why this deliverable aimed to design or identify processes and raw materials that are potentially recyclable, reusable or reusable.

![Some team members at a stoplight asking for money to support the team.](https://static.igem.wiki/teams/4147/wiki/notebook/july25-31.png)

**## **Week of August 1st to 7th****

1st: The human area had the opportunity to make a visit to the Foster Home “Unidas por Amor”. There we gave an introduction into science and synthetic biology to kids around 10-18 years old. Later we got the chance to give them our comic story and invited them to paint it. We rad it with them and had a session of comprehensive questions.

3rd: We got a chance to speak about our project at a press conference in the city of Cuauhtemoc. Our interview was broadcasted in several news media from the city. Also, this was the day our distribution kit arrived!

![Distribution kit.](https://static.igem.wiki/teams/4147/wiki/notebook/aug1-7.png)

![Picture on the right is Elisa al the lab holding the kit. Picture on the left is Alessandra at the lab holding the kit.](https://static.igem.wiki/teams/4147/wiki/notebook/aug1-7-1.png)

4th: Tec-Chihuahua in Clubes de Ciencia! Our PI made a presentation about the competition and our team at the Science Coffee.

![PI in a classroom giving a presentation about the iGEM team.](https://static.igem.wiki/teams/4147/wiki/notebook/aug1-7-2.png)

The PhD Estefanía Ramírez has a wide experience in Phytophthora capsici. She helped us fully understand the way this oomycete attacks, and why it is classified as a strong pathogen. This information was useful to shape or find a way to strengthen the solution we are trying to develop for this infection.

5th: The university Tecnologico de Monterrey makes sure new students feel comfortable their first days of classes and for that they make this event called HiTEC. In this event the new students get to know each other through ice breaker activities. We participated in the event with an activity that resembled a ‘laser’ like situation. We used yarn as the lasers and the students were meant to move from one side to another without touching the ‘lasers’. As they went from one side to the other, they needed to pick a biobrick to make a construction.

We reached the Institute of Mexican Sign Language in the city of Chihuahua. We believe science should be accessible for everyone, but not everyone makes their content thinking of all the people that may have trouble accessing it. So we thought of making our videos accessible for deaf people. The Institute was really kind in helping us translate our videos.

The wet Lab team continued trying to induce the expression of the GAFP-1 protein assembled weeks before. In order to see if the induction was successful, they started doing SDS-PAGE.

The entrepreneurship area works with basic principles. After having reflected on what is needed to carry out this, we learned the basic principles that govern an entrepreneurship ecosystem.

Likewise it has been made the feasible product deliverable After having determined the preliminary hypotheses for the solution following the methodology of the pre-incubation program.

![Chuy and Arlen at a science cafe.](https://static.igem.wiki/teams/4147/wiki/notebook/aug1-7-3.png)

The model team had their first results by solving the system of ODEs in Matlab. We later had a meeting with our instructor to analyze the behavior of our graphics.

**## **Week of August 8th to 14th****

This week the Wet Lab team received the IDT sequences, so they ran PCR of selected fragments in order to amplify them and do digestion protocol. The results were not favorable :(

![IDT package.](https://static.igem.wiki/teams/4147/wiki/notebook/aug8-14.png)

The entrepreneurial team began with the design of a landing page for the future implementation of our product as well as the plans and models for the future biopesticide marketing company.

The entrepreneurship area works Business model hypothesis like the preliminary hypothesis once having a broader picture of all the implications that a startup entails for the development and production of biopesticides through a business model canvas, the following aspects necessary to carry out this achievable business were reflected.

This week the modeling team started working on the industrial scale-up of our process. We also ran into some problems with Symbio, so we kept working to try and solve them.

11th: We had the opportunity to visit a girls only foster home to teach them basic science concepts and we reinforced them with some activities. They learned about what a cell is and the instruction book (DNA) each of them has to know what they should do. We also gave each girl our comic for them to color and that way they learned about chilli wilt.

**## **Week of August 15th to 21th****

15th: We talked to SEECH (Servicios Educativos del Estado de Chihuahua), which is a governmental organization that looks after educational services in the state of Chihuahua. They were kind to help us distribute the comic and manuals that were previously translated to two different ethnic languages to said ethnic groups.

19th: Our shooting for the promotion video started.

![Team members at a chilli field.](https://static.igem.wiki/teams/4147/wiki/notebook/aug15-21.png)

The wet lab team worked on trying to amplify and reamplify some sequences. They found out the primers were amplifying when they made a test with sequences from previous years, so that way they could identify that the DNA from IDT was degraded.

The model team didn’t have much luck with Symbio, so unfortunately they left it behind. On the other hand, they presented some results of the industrial scale-up.

![Industrial scale up in a computer software, Super Pro Designer.](https://static.igem.wiki/teams/4147/wiki/notebook/aug15-21-1.png)

The entrepreneurship area carried out the format of the label with the requirements of the agencies of the federal commission for the protection of health risks and the environmental protection agency. The trademark registration process for the product. This with the purpose of giving formality to the legal process as well as having the sustenance in case of being falsified.

![First attempt at our product's label.](https://static.igem.wiki/teams/4147/wiki/notebook/aug15-21-2.png)

Likewise, a photo session was also held for the landing page, presentations and social networks.

![Saghir possing at a chilli field with aour product in hand.](https://static.igem.wiki/teams/4147/wiki/notebook/aug15-21-3.png)

19th: We visited the Secretaria de Desarrollo Rural, which is the gubernamental institution that oversees the development of the rural areas of Chihuahua. They were kind enough to explain to us the importance of the production of chilli and how we should promote a culture of prevention of pathogens to agricultors to preserve their crops. Especially with the pathogen we are trying to attack, the best way to avoid it is through prevention.

![Team members sat down at the Secretaria de Desarrollo Rural.](https://static.igem.wiki/teams/4147/wiki/notebook/aug15-21-4.png)

**## **Week of August 22th to 28th****

23rd: We had a meeting with the faculty of forestry and agricultural sciences where we talked about presenting our conference of the introduction to synthetic biology and distribution of our manual. They invited us to make this a requirement in the institution so that everyone has to take the course.

![Human Practices team members in a auditorium at the faculty of forestry and agricultural sciences. A lot of attendees.](https://static.igem.wiki/teams/4147/wiki/notebook/aug22-28.png)

24th: The Human area presented a conference in collaboration with the scientific diffusion network Divulgare where 2 team members performed the process of DNA extraction in strawberries with home materials. This was shown via zoom where we had 32 expectators.

![Picture on the left is the zoom meeting where the conference was held. The picture on the right is the poster for the event.](https://static.igem.wiki/teams/4147/wiki/notebook/aug22-28-1.png)

Also we finished the comic book compendium in english, meaning all of the collaborators material was unified in a single document.

25th: We had a meeting with the Zootecnia Faculty where we talked about giving a conference about our project and synthetic biology and the distribution of our manual.

Entrepreneurship: Stella Orion invited us to practice our pitch and gave us feedback on our product, our company and our presentation.

![The entrepreneurship team catching up the rest of the team on the progress of their area.](https://static.igem.wiki/teams/4147/wiki/notebook/aug22-28-2.png)

**## **Week of August 29th to September 4th****

This week the model team made a sensitivity analysis of their system of ODEs.

![Morris screening graphs.](https://static.igem.wiki/teams/4147/wiki/notebook/aug29-4.png)

30th: We attended “Agronomy” (Agronomía), an event in which we held a conference about our project: Agrocapsi and an introduction to iGEM and synthetic biology. The public (95 people) were experts in phytopathology and agriculture, they gave us feedback and asked a lot of questions. We also distributed our manual.

![Human practice's team members giving a conference at the agronomy university.](https://static.igem.wiki/teams/4147/wiki/notebook/aug29-4-1.png)

We gave a presentation of our project in “Faciatec” (Faculty of Agrotechnological Sciences). Around 80 people attended and we received a lot of feedback and asked many questions.

We attended the elementary school ” Colegio Pierre Faure”, where we gave a small class about science and synthetic biology and an activity regarding molecular biology.

2nd: We submitted our work at the 2022 Agrigenomics for Food and Health: Connecting Engineering, Science and Industry. For this congress, we made a scientific poster to spread the work we have done.

![Our scientific poster at the Agrigenomic's event.](https://static.igem.wiki/teams/4147/wiki/notebook/aug29-4-2.png)

**## **Week of September 5th to 11th****

The model team had a reunion with team Tec-Monterrey to analyze the characteristics of our siRNA sequence.

![Zoom meeting with Tec-Monterrey's team.](https://static.igem.wiki/teams/4147/wiki/notebook/sept5-11.png)

5th: Our instructor Cesar broke a watermelon with his legs. This was clearly a crucial event in the development of our project.

![Cesar in a football field with a watermelon in beetween his legs.](https://static.igem.wiki/teams/4147/wiki/notebook/sept5-11-1.png)

6th: The Human team had a meeting with Zootecnia and Ecology Faculty where Wilting and our product development was exposed, focusing on the environmental impact that pesticides have and how this situation can be diminished with the use of Synthetic Biology.

8th: The Human team assisted Agrogreen to perform an interview with Judith Cambia on the Certification Norms required for those who cultivate to commerce it or to people like us who are trying to create a product.

10th: Through zoom we had the agricultural workshop we planned alongside UAM’s team. It was in the morning and lasted around 2 hours. We had a lot of participation from the audience and asked interesting questions about current problematics and the use of new technologies to solve them.

11th: Astronomia (astronomy science group) invited us to an event they planned. They had a ‘Watch the moon and the planets’ night where they gave us a space to talk to the attendees about how synthetic biology can be used for space.

**## **Week of September 12th to 18th****

The model team encountered some troubles in the solution of their system of ODEs. This was due to some parameters being wrong, so they started correcting that right away. They also analyzed the behavior of the system under different conditions.

The wet lab team received some sequences from Twist so finally could work on digestion and ligation of the interest parts of our project. At this point the ligation protocol was already standardized to the better conditions of enzyme and ratio.

12th: We had a meeting with ICHEA (Chihuahua’s Institute for Adult’s Education) where they helped us translate our comic and manual to Rarámuri (indigenous language in Chihuahua). In this meeting we made sure to explain the intention of the manual, the objective and general contents of it.

14th: We attended a primary school named Carmen Serdan in Delicias City in Chihuahua State. We had a small 30 minute talk with all the elementary kids, 12 groups total. The small presentation was about basic concepts of science such as what is a microorganism and what is DNA. That way we could give examples of how these are used in synthetic biology to help humanity out.

22nd: CBTA90 is a high school in Cuauhtemoc City where kids can graduate with a technician degree. We had the opportunity with 3rd and 5th semester biotechnology students from that school to make a mini iGEM. All this with the purpose of awakening the interest in biology applications and to show them that their ideas could actually be great future solutions.

23rd: In Camargo City there is this news broadcast called Impacto Noticias, they kindly gave us about 10 minutes airtime to talk about our project and the competition. This interview highlighted the importance of finding a solution to chilli wilt due to its impact nationally. The clip of the interview can be found on their Facebook page.

**## **Week of September 19th to 25th****

After making some corrections, the model team had a reunion with PhD Roberto to analyze the new behavior of the system. They also had a reunion with instructor Silveyra to review the sensitivity analysis from a statistical point of view.

The wet lab team received the rest of the sequences from Twist. The vectors were pretty good at transforming our strain. We finally had A LOT of colonies.

21st: We collected several books to give away to those in need. We went to a foster home we had gone to before to give them these books.

24th: In order to receive feedback from producers, we had this event we named Agrohack so that we could talk about our project. After we explained the technicalities of our solution that gave us suggestions and were really glad that some of the previous comments they made were taken into consideration. We also had Dr. Juan Aguirre talk about his work and investigation in agricultural topics.

24th: A public high school gave us the opportunity of making our intelligences rally on saturday with a couple of their groups.

**## **Week of September 26th to October 2nd****

The wet lab team started to induce the sequences they transformed from vectors. They also set up an expression kinetic for the model team to recover information of the promoters and include that data into our mathematical model. The data were not reliable :(

The modeling and entrepreneurship team worked on scaling up our process.

**## **Week of October 3rd to 9th****

6th: As our mini iGEM activity proved to be a success, they gave us an opportunity to do it once again in our school’s high school. These kids also had some kind of biotechnological background, cause in 5th semester they have a topic class where they can choose biotechnology.

8th: We had a sleepover at school to finish our wiki.

**## **Week of October 10th to 12th****

It has been months of hard work. We are glad you took the time to read our notebook.

Thank you so much!

![iRNAldo is very glad because you read our whole notebook.](https://static.igem.wiki/teams/4147/wiki/parts/agradecimientos.png)

Content

Content

[**Go to Parts**

![](https://static.igem.wiki/teams/4147/wiki/bus.png)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)

{% 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 id %} id="portada\_engineering" {% endblock %}
{% block page\_content %}

[Introduction](#introduction)
[Math model](#Math12)
[Bacterial transformation](#1_1)
[Peptide expression](#2_1)
[siRNA](#6iteration)
[References](#references)

**#### Overview**

Welcome to the **engineering success** page!

Here we describe the development of our project: How, as we progressed, we discovered new challenges which forced us to find new solutions and initiate new engineering cycles. In this section you will see that the development of our project was not a linear process as presented in our [Results](https://2022.igem.wiki/tec-chihuahua/results).

![](https://static.igem.wiki/teams/4147/wiki/education/irnaldos/eng-success.png)

# **INTRODUCTION**

To find a solution to a problem through synthetic biology, it is necessary to follow iterations of the DBTL (Design, Build, Test, Learn) cycle. This cycle generates a bridge between the problem with science and the technologies to be implemented ([Lawson *et al*., 2019](r4)).

With Agrocapsi, we aimed to develop a biofungicide that would help prevent wilt. Nonetheless, we wanted a product without the adverse effects that current agrochemicals have. Behind the obtained results there was a lot of hard work that went through key decision-making moments that shaped the project to what it is today.

In synthetic biology these iterations of the DBTL cycle are very common. No matter how carefully we design the constructs, the experiments do not always work as planned. Throughout the project we have performed a lot of DBTL cycles. Below are some of the most significant ones for our project. **Click on each of them to learn more.**

## **math model**

[![](https://static.igem.wiki/teams/4147/wiki/btn1.png)](#Math12)

## **BACTERIAL transformation**

[![](https://static.igem.wiki/teams/4147/wiki/engineering/1-1.png)](#1_1)

[![](https://static.igem.wiki/teams/4147/wiki/engineering/1-2.png)](#1_2)

[![](https://static.igem.wiki/teams/4147/wiki/engineering/1-3.png)](#1_3)

## **PEPTIDE EXPRESSION**

[![](https://static.igem.wiki/teams/4147/wiki/design/2-1.png)](#2_1)

[![](https://static.igem.wiki/teams/4147/wiki/design/2-2.png)](#2_2)

## **siRNA**

[![](https://static.igem.wiki/teams/4147/wiki/engineering/sirna-1.png)](#siRNA_1)

[![](https://static.igem.wiki/teams/4147/wiki/engineering/sirna-2.png)](#siRNA_2)

# **MATH MODEL**

## **1st iteration: Redesign of our mathematical model**

[![](https://static.igem.wiki/teams/4147/wiki/engineering/1st.png)](#podcastImpact)

![](https://static.igem.wiki/teams/4147/wiki/education/irnaldos/t-attributions.png)

![](https://static.igem.wiki/teams/4147/wiki/engineering/design-b38600.png)

**Design:**

Through our modeling, we sought to determine the optimal concentrations of IPTG and an appropriate promoter for our expression system. This way, we could achieve our goal of optimizing our production process. Thus, we decided to model the gene expression of an IPTG-inducible and LacI-repressible system in our *E. coli* BL21(DE3) chassis.

**Build:**

![](https://static.igem.wiki/teams/4147/wiki/engineering/build-b38600.png)

Once we had the idea, we got down to work. We made a step-by-step diagram showing the two possible scenarios of our expression system: with and without IPTG in the medium.
Once we had the diagram we defined the assumptions of our model and described each of the biochemical reaction, and finally we constructed a system of equations based on the law of mass action and mass balance.

In addition, we defined parameters based on a literature review.

![](https://static.igem.wiki/teams/4147/wiki/engineering/test-b38600.png)

**Test:**

We solved the ordinary differential equations (ODEs) and ran computer simulations to observe the behavior of our plots.

However, the results were not quite as expected. We observed that our system did not behave like in our first laboratory experiments. Our instructor, Ph.D. Roberto Olivares, mentioned that there were 3 aspects that could be causing an inadequate behavior:

![](https://static.igem.wiki/teams/4147/wiki/engineering/intento2-con-ajuste-de-variables.jpg)

**Figure 1.**First solution to the first model we created.

1. Errors when writing the equations in our code (since some parentheses or signs were different from the equations we had previously designed).
2. Errors in the units of our parameters.
3. Errors in the mass balances.

So we patiently reviewed each of the points recommended by our instructor.

**Learn:**

![](https://static.igem.wiki/teams/4147/wiki/engineering/learn-b38600.png)

We learned that there were steps we were not considering in the mass balances. This allowed us to redesign our system of ODEs by restarting the engineering cycle.

This led us to perform many more DBTL cycles. It was not only to change the mass balances but also to look for other parameters that had similar experimental conditions to ours.

This allowed us to build the model that we present in the [Model](https://2022.igem.wiki/tec-chihuahua/model) page.

# **BACTERIAL TRANSFORMATION**

## **1st iteration: First ligation and cloning attempt**

![](https://static.igem.wiki/teams/4147/wiki/engineering/design-b38600.png)

**Design:**

At the begining, the first step was defining the antimicrobial molecules to use. We selected the genes of interest, an osmotin and a dermaseptin, as well as the biobricks needed to build our expression insert. The assembly design was first performed *in silico* in SnapGene by adding the standard biobricks prefix and suffix and making ligations between promoters [BBa\_R0010, BBa\_R0011], RBS [BBa\_B0032], the coding sequence of interest and the double terminator [BBa\_ B0015], as shown in Figure 2. Then, the sequences were optimized for *E. coli* and finally requested to IDT as part of the sponsorship.

![](https://static.igem.wiki/teams/4147/wiki/engineering/constructos.png)

**Figure 2.** Examples of designed sequences. A) Expression cassette for PcOSM peptide with DsbA. B) Expression cassette for (CBD)2-DrsB1 peptide with DsbA. C) Expression cassette for PcOSM peptide. D) Expression cassette for (CBD)2-DrsB1 peptide.

**Build:**

![](https://static.igem.wiki/teams/4147/wiki/engineering/build-b38600.png)

After receiving the sequences from IDT, we performed the proper digestion of the fragments using EcoRI and PstI as advised by the provider. The linearized plasmid pSB1C3 that we had from earlier editions was likewise digested. A LacI regulated promoter BioBrick [BBa\_R0010] plasmid was simultaneously digested as a control as demonstrated in Figure 3. With this gel we verified the correct activity of our enzymes and digestion conditions. We then proceeded to perform the ligation protocol of our inserts with the pSB1C3 vector.

![](https://static.igem.wiki/teams/4147/wiki/engineering/f1.jpg)

**Figure 3.** LacI regulated promoter BioBrick [BBa\_R0010] digested with different enzymes in a 0.8% agarose gel. Expected band size: 2268 bp. A) QuickLoad Purple Plus DNA Ladder. B) LacI regulated promoter BioBrick uncutted. C) BBa\_R0010 digested with EcoRI. D) BBa\_R0010 digested with XbaI. E) BBa\_R0010 digested with PstI. F) BBa\_R0010 digested with SpeI (2017). G) BBa\_R0010 digested with SpeI (2018).

![](https://static.igem.wiki/teams/4147/wiki/engineering/test-b38600.png)

**Test:**

The ligation product was transformed into *E. coli* BL21 (DE3) by heat shock and then inoculated into medium containing chloramphenicol. Unfortunately, we did not obtain transformed clones with any of our constructs, so we concluded that something was wrong with our protocols or design.

**Learn:**

![](https://static.igem.wiki/teams/4147/wiki/engineering/learn-b38600.png)

After analyzing the possible sources of error we realized that for efficient cleavage, our insert needed to have extra base pairs at the end of the recognition site. This need was corroborated with the gel shown in **Figure 3**, where defined bands could be seen due to the cuts made by the enzymes in a sequence that had the required base pairs flanking the recognition site. In other words, the LacI regulated promoter BioBrick in **Figure 3** could be digested correctly because it was a circular construct. However, the fragments of interest were linear inserts whose ends were flanked only by the suffix and prefix. Having said that, we had to go through a new engineering cycle to prove this new hypothesis.

## **2nd iteration: Amplification of our inserts**

![](https://static.igem.wiki/teams/4147/wiki/engineering/design-40b0a6.png)

**Design:**

Following the learnings from the previous cycle, we carefully analyzed the manufacturer's considerations for each enzyme to know the number of base pairs needed for the cleavage. After evaluating the options, designing and synthesizing primers for a PCR amplification was the most viable option for us. The primers were first created *in silico* in SnapGene to hybridize to the prefix and suffix of each part. Once we obtained these sequences, we had them synthesized in order to continue our work.

**Build:**

![](https://static.igem.wiki/teams/4147/wiki/engineering/build-40b0a6.png)

It was important to develop the protocol taking into account the melting temperature (Tm) of our primers as well as the number of cycles, phase temperatures, and timings to perform PCR of the sequences we already had.

At this point, the amount of insert left to work was low, due to our previous attempts to ligate our IDT sequences. This didn't allow us to commit any errors. As a result, we decided to test our primers using some sequences that were left over from from previous teams. The prefix and suffix that were used to construct these fragments, along with the fact that our primers were made to hybridize to these sequences, should theoretically lead them to amplify. This amplification had the result depicted in Figure 4.

![](https://static.igem.wiki/teams/4147/wiki/engineering/f3.jpg)

**Figure 4.** Sequences from prior teams, abaecin and defensin in 0.8% agarose gel after amplification with prefix and sufix specific primers. A) QuickLoad Purple Plus DNA Ladder. B) Abaecin sequence (361 pb) at Ta= 50° C. C) Defensin sequence (415 pb) at Ta= 55° C. D) Abaecin sequence (361 pb) at Ta= 55° C. E) Defensin sequence (415 pb)at Ta= 55° C.

![](https://static.igem.wiki/teams/4147/wiki/engineering/test-40b0a6.png)

**Test:**

We carried out PCRs with our fragments under the same conditions after confirming that our primers were effective and amplifying sequences with prefixes and suffixes. However, there was no banding or amplification visible in the electrophoresis gels Figure 5.

![](https://static.igem.wiki/teams/4147/wiki/engineering/f4.jpg)

**Figure 5.** Project sequences PelB-(CBD)2-DrsB1 with DsbA, (CBD)2-DrsB1, DrsB1 construct with LacI Promoter, and PcOSM construct with DsbA in 0.8% agarose gel after amplification with prefix and sufix specific primers. A) QuickLoad Purple Plus DNA Ladder. B) PelB-(CBD)2-DrsB1 construct with DsbA (1738 bp) at Ta= 50 °C. C) Expression construct for (CBD)2-DrsB1 (1075 bp) at Ta= 50 °C. D) DrsB1 Construct with LacI Promoter (481 bp) at Ta= 50° C. E) PcOSM Construct with DsbA (1738 bp) at Ta= 50° C. F) QuickLoad Purple Plus DNA Ladder.
G) PelB-(CBD)2-DrsB1 Construct with DsbA at Ta= 55° C. H) Expression construct for (CBD)2-DrsB1 at Ta= 55° C. I) DrsB1 Construct with LacI Promoter at Ta= 55° C. J) PcOSM Construct with DsbA at Ta= 55° C.

**Learn:**

![](https://static.igem.wiki/teams/4147/wiki/engineering/learn-40b0a6.png)

We could tell from the gel's results that the DNA was not in great condition, which is why none of the fragments were amplified. The fact that they were huge fragments, as IDT said, made the PCR challenging to carry out. We notified the corporation about these difficulties, justifying our hypothesis, and they agreed to replace the damaged sequences.

However, in our region it is common to wait quite a long time for synthesis products and molecular reagents to arrive, since they all have to be imported and are usually held up in Customs and Border Protection for some time. In this edition, it was particularly challenging as the waiting times were up to months. As a result, we had to reconsider our alternatives.

## **3rd iteration: Ordering new inserts**

![](https://static.igem.wiki/teams/4147/wiki/engineering/d-000.png)

**Design:**

Although IDT very kindly accepted the replacement of the damaged DNA, due to the lack of time, we considered the sponsorship of Twist Bioscience for a new order. Before placing the order, the constructs were modified and optimized again. We added adapters to both ends, which provided the additional nucleotides required for effective digestion. This allowed us to avoid a PCR amplification and eliminate the risk of obtaining sequences with a mutation with our low fildelity polymerase. At this time, we could established contact with the parcel delivery company, which accelerated this subsequent shipments a bit.

**Build:**

![](https://static.igem.wiki/teams/4147/wiki/engineering/b-000.png)

We were able to digest the vector and inserts with EcoRI and PstI as soon as the sequences with adapters arrived. But then, the ligation technique planned was not working either, so we had to create a shorter engineering cycle. We experimented with altering the insert and vector ratios, beginning with the molar ratios recommended by the manufacturer (3:1, 5:1, and 7:1). None of the ratios worked. We subsequently decided to carry out this protocol's assembly using mass ratios and plasmid dephosphorylation. In one of the ligation experiments, we did see colonies after transformation. Thus we deduced that the ideal circumstances were with the vector dephosphorylated at a 3:1 ratio of insert and plasmid, respectively.

![](https://static.igem.wiki/teams/4147/wiki/engineering/test-000.png)

**Test:**

The ligation product was transformed into *E. coli* BL21 (DE3) by heat shock and then inoculated into medium containing kanamycin. This time, we were able to produce positive clones. When this procedure was repeated for a subsequent ligation, we likewise got favorable outcomes.

**Learn:**

![](https://static.igem.wiki/teams/4147/wiki/engineering/l-000.png)

With the results achieved, all the relevant methods, including digestion, ligation, and the transformation of the *E. coli* strain, could be standardized. Finally, we used colony PCR to confirm that these steps were proper and that our inserts were in fact present in the bacteria. The findings are depicted in Figure 6.

![](https://static.igem.wiki/teams/4147/wiki/figurano-6-engineering.png)

**Figure 6.** Colony PCR from clones with mCherry (RFP) construct with LacI promoter, (CBD)2-DrsB1 construct, PcOSM construct with LacI promoter, and mCherry (RFP) construct with lambda pL hybrid promoter in 0.8% agarose gel. A) QuickLoad Purple Plus DNA Ladder. B) mCherry (RFP) construct with LacI regulated promoter (1137 bp) at Ta= 50° C. C)Expression construct for (CBD)2-DrsB1 (1134 bp) at Ta= 50° C. D) PcOSM Construct with LacI regulated promoter (1134) at Ta= 50° C. E) mCherry (RFP) construct with LacI regulated, lambda pL hybrid promoter (992 bp) at Ta= 50° C. F) PcOSM construct with DsbA (1797 bp) at Ta= 50° C.

We have confirmed that now we have transformed cells with our expression vectors!

# **PEPTIDE EXPRESSION**

## **4th iteration: Inducing the expression of our peptides**

![](https://static.igem.wiki/teams/4147/wiki/engineering/design-b38600.png)

**Design:**

Once we obtained successful transformations, we used isopropyl-D-1-thiogalactopyranoside (IPTG) to induce expression, since both promoters selected (regulated LacI promoter and lambda pL promoter hybrid) were inducible by it. To determine the best induction concentration, we created a protocol with different concentrations of IPTG. To visualize the induction results, an SDS-PAGE protocol was followed. The weights of our proteins are 4 kDa for DrsB1 (the existing part), 25 kDa for improved DrsB1, and 25.6 kDa for PcOSM with LacI promoter.

**Build:**

![](https://static.igem.wiki/teams/4147/wiki/engineering/build-b38600.png)

The induction was carried out using a kinetic at IPTG concentrations of 0 mM, 0.1 mM, 0.5 mM, and 1 mM with 6 hours of incubation. This would enable us to determine the level of IPTG at which the highest synthesis of our target molecules will occur. Additionally, an SDS-PAGE methodology that was appropriate for the protein weight was developed, and it was decided to create acrylamide gels made up of concentrator and separator gels at 6% and 12%, respectively.

![](https://static.igem.wiki/teams/4147/wiki/engineering/test-b38600.png)

**Test:**

Centrifugation was used to separate the soluble and insoluble fractions from each culture of *E. coli* BL21 (DE3) that had been transformed and induced at the appropriate IPTG concentration. Increasing concentrations of each fraction were put onto SDS-PAGE gels. To compare the banding pattern, an untransformed induced *E. coli* BL21 (DE3) control was loaded. Mercaptoethanol and a brilliant blue-based loading buffer were used to prep the samples. For 20 minutes at 80 V and 35 minutes at 180 V, these gels were tested. As demonstrated in Figure 7, it was easy to see that the proteins of interest were absent from both soluble and insoluble fractions after staining and destaining. The lack of a difference in the banding pattern between the control sample and the relevant samples in the targeted weight region allowed for the deduction that the protein was absent.

![](https://static.igem.wiki/teams/4147/wiki/design/f6.jpg)

**Figure 7.** SDS-PAGE of soluble and insoluble protein fractions obtained from a culture media induced by different IPTG concentrations for 6 hours. A) Page Ruler ™ Low Range Unstained Protein Ladder.B) *E. coli* BL21 Induced with IPTG 1 mM C) DrsB1 existing induced with IPTG 0 mM soluble fraction. D) DrsB1 existing induced with IPTG 0 mM insoluble fraction. E) DrsB1 existing induced with IPTG 0.1 mM soluble fraction. F) DrsB1 existing induced with IPTG 0.1 mM insoluble fraction. G) DrsB1 existing induced with IPTG 0.5 mM soluble fraction
H) DrsB1 existing induced with IPTG 0.5 mM insoluble fraction. I) DrsB1 existing induced with IPTG 1 mM soluble fraction. J) DrsB1 existing induced with IPTG 1 mM insoluble fraction.

**Learn:**

![](https://static.igem.wiki/teams/4147/wiki/engineering/learn-b38600.png)

Different hypotheses were considered when unfavorable results with induction were observed. A possible defect in the design of the constructs, particularly in the promoter region, was one of the things we thought might have played an important role. Also, the IPTG used could not be in the best condition due to the expiration date, which may have contributed to the failure to promote induction.

There are many sources of error in having induced these sequences for the first time. It is possible that the experiment was not performed at the proper concentrations and/or time, or that the protocol that was established suffered from an error in execution. Therefore, it was necessary to introduce a new engineering cycle.

## **5th iteration: Checking our expression cassettes with a fluorescent protein**

![](https://static.igem.wiki/teams/4147/wiki/engineering/design-40b0a6.png)

**Design:**

To confirm which of our hypotheses was true and to find the reason why our protein did not seem to be expressed, we decided to reduce the variables to make the analysis easier.

We set up induction kinetics with expression cassettes identical to the ones we used to express our peptides. Only this time we replaced our peptides with a flourescent protein: mCherry.

Likewise, we used as a control a vector we had in the laboratory. This vector consisted of a pET28 expressing another mCherry protein. This construct, despite having a different promoter, was also inducible by IPTG like ours, so it could either confirm or refute some of our hypotheses. However, it was decided to redesign the protocol to collect samples at different induction times with varying inducer concentrations.

**Build:**

![](https://static.igem.wiki/teams/4147/wiki/engineering/build-40b0a6.png)

Colonies of *E. coli* BL21 (DE3) previously transformed with the constructs of interest were grown until they reached an OD600 of 0.4 to 0.6. Once this OD600 was reached, IPTG was added at different concentrations to start induction (0 mM, 0.1 mM, 0.5 mM and 1 mM).

This time we used a different IPTG stock than the one used in the previous cycle. We also took samples at different times (1h, 2h, 8h) to determine concentration and optimal induction time.

![](https://static.igem.wiki/teams/4147/wiki/engineering/test-40b0a6.png)

**Test:**

After induction, an SDS-PAGE was set up. Specifically, the soluble fraction of the transformed bacteria was loaded. Untransformed *E. coli* BL21 (DE3) strain with and without induction was used as a control. The gels were run at 80 V for 20 min, and at 180 V for 35 min. The results of the gels are shown in Figures 8 and 9. The expected protein was observed, but no induction was noticed.

![](https://static.igem.wiki/teams/4147/wiki/design/f7.jpg)

**Figure 8.** A) Page Ruler ™ Low Range Unstained Protein Ladder. B) BL21 (DE3) without induction. C) BL21 (DE3) induced 1 mM. D) mCherry control with 0 mM 1 hour. E) mCherry control 0.1 mM 1 hour F) mCherry control 0.5 mM 1 hour. G) mCherry control 1 mM 1 hour. H) mCherry control 0 mM 2 hours. I) mCherry control 0.1 mM 2 hours. J) mCherry control 0.5 mM 2 hours.

![](https://static.igem.wiki/teams/4147/wiki/design/f8.jpg)

**Figure 9.** Polyacrylamide gel 12% for visualization of the SDS-PAGE results of the mCherry control and mCherry-Hybrid promoter construct. The expected band size of the mCherry control is 31.1 kDa and 26.7kDa for mCherry-Hybrid promoter. A) Page Ruler ™ Low Range Unstained Protein Ladder. B) mCherry control 0 mM 8 hours. C) mCherry control 0.1 mM 8 hours. D) mCherry control 0.5 mM 8 hours. E) mCherry control 1 mM 8 hours. F) mCherry-Hybrid promoter 0 mM 1 hour. G) mCherry-Hybrid promoter 0.1 mM 1 hour. H) mCherry-Hybrid promoter 0.5 mM 1 hour. I) mCherry-Hybrid promoter 1 mM 1 hour. J) mCherry-Hybrid promoter 0 mM 2 hours.

**Learn:**

![](https://static.igem.wiki/teams/4147/wiki/engineering/learn-40b0a6.png)

Although the banding pattern on the gels indicated the presence of protein, we can deduce that in both cases we obtained partially positive results because we could see protein expression but not regulation through IPTG induction. This was determined by comparing the bands of each construct in non-induced and induced samples (Figures 8 and 9). Although the results were not as expected, we can rescue one positive aspect of this experiment: we were able to observe the activity (although constitutive) of the promoters. Furthermore, as a second benefit, these experiments allowed for the reduction of variables that could be sources of error. However, we had to evaluate the factors that still remained to be considered.

The possibility that the induction protocol did not work was raised because it was executed at different times by all members of the laboratory area. If it had been assembled by only one person, the possibility of human error would have been reduced. As a result, this is something we would change in a future assembly. On the other hand, these results were consistent with those obtained from a fluorescence assay. The data obtained from this assay, however, were not completely reliable due to a high variance caused by several factors, including the type of plate used, which was neither black nor opaque, as well as the reading configuration and direction. Furthermore, we consider performing this test directly in the location where the reading will be performed in future assemblies, because the transport of the plates could have been influenced by some mishandling of the plates during the journey.

We would also experiment with different induction temperatures to see if this played a role in the results. It has been shown that lowering the temperature at which the protein is expressed may be the most simple and successful method of obtaining protein (Duong-Ly, 2014). That's why in the future experiments we will try induction from 15–28 °C, a range that has been tested and used successfully (Kang *et al.*, 2003).

# **siRNA**

## **6th iteration: siRNA expression vector construction**

![](https://static.igem.wiki/teams/4147/wiki/engineering/design-b38600.png)

**Design:**

For siRNA design we performed a literature search to find candidate genes involved in the pathogenicity or viability of the oomycete. One of the strongest candidate genes to be silenced were those encoding for the RXLR effector proteins of *P. capsici*. These proteins suppress the plant immune system, and are one of the reasons why *P. capsici* is such an aggressive pathogen.

Once we selected the gene we began to design the sequence of the siRNA we planned to produce. For this we used the online tool E-RNAi ([Horn & Boutros, 2010](#r3)).

For siRNA selection we followed the following considerations [(Fakhr *et al.,* 2016):](#R2)

* The first nucleotide of the siRNA sequence can either be an A or a G.
* Choose sequences with low GC content.
* Avoid 5' and 3'UTRs.
* Select a ~21 nt sequences in the target mRNA.

Also, in [collaboration](https://2022.igem.wiki/tec-chihuahua/collaborations) with the Tec-Monterrey team, we performed an analysis of the **thermodynamic properties** of our sequence. For this we used a tool they developed to evaluate the relative efficiency of siRNAs, where the worst siRNAs have an efficiency of 0 and the best ones have an efficiency close to 100. Our siRNA showed an efficiency of 93%. You can read more on the [siRNA](http://parts.igem.org/Part%3ABBa_K4147016) registration page.

![](https://static.igem.wiki/teams/4147/wiki/engineering/modelo-matem-tico-3.png)

**Figure 10.** Relative hibridization efficiency of possible siRNAs that target RXLR gene of *P. capsici*. Our designed siRNA showed a Grelative efficiency of hibridization of 93%.

We then analyzed homology with other species using BLAST, thus maintaining the biosafety of other living organisms.

**Build:**

![](https://static.igem.wiki/teams/4147/wiki/engineering/build-b38600.png)

In the future, we plan to assemble the Up and Down sequences of the expression cassette. For this purpose, a 3A assembly will be performed: the Up part will be cut with EcoRI and SpeI enzymes. The Down part will be cut with XbaI and PstI enzymes. And the vector where they will be inserted will be cut with EcoRI and PstI enzymes (Figure 11).

![](https://static.igem.wiki/teams/4147/wiki/sirna-final.png)

**Figure 11.** Expression cassette assembly method for siRNA production. Inspired by the Ecuador 2021 team..

*E. coli* HT115 cells will be transformed with this vector.

![](https://static.igem.wiki/teams/4147/wiki/engineering/test-b38600.png)

**Test:**

To corroborate the correct transformation of the cells, a PCR of the colonies grown in the medium with antibiotics will be performed. The primers shown below will be used for this purpose.

| PRIMER 1 | GTTTCTTCCTGCAGCGGCCGCTACTAG |
| --- | --- |
| PRIMER 2 | GTTTCTTCGAATTCGCGGCCGCTTCTA |

It is expected to have a gel as shown in Figure 12 to confirm the presence of the inserts.

![](https://static.igem.wiki/teams/4147/wiki/pcr-sirna.png)

**Figure 12.** Simulation of a 1% agarose gel with the 1 kb Plus DNA Ladder Molecular Weight Marker. Colony PCR amplicon is observed confirming the presence of the insert.

**Learn:**

![](https://static.igem.wiki/teams/4147/wiki/engineering/learn-b38600.png)

Once the transformed colonies are obtained, we can proceed to the production of the molecules of interest.
We know that it will most likely take many more iterations to complete this cycle.

## **7th iteration: Efficiency of siRNA to reduce pathogenicity**

![](https://static.igem.wiki/teams/4147/wiki/engineering/design-40b0a6.png)

**Design:**

To prove that our siRNA works we plan to perform an inhibition assay. PhD. Estefanía Ramírez, recommended us to perform our inhibition assays *in vitro* growing chilli plants in V8 liquid medium and placing an inoculum of *P. capsici* so that the pathogen is active.
Afterwards, we would place the different RNAi treatments on that medium.

The experimental design is shown in Figure 13.

![](https://static.igem.wiki/teams/4147/wiki/engineering/2-untitled-slide-4.png)

**Figure 13.** Experimental design of assay testing effect of siRNA on pathogenicity of *P. capsici*. Concentrations were defined based on previous experiments ([Cheng *et al.,* 2022](#R1)).

**Build:**

![](https://static.igem.wiki/teams/4147/wiki/engineering/build-40b0a6.png)

Once the experiment was set up, disease lesions will be measured twice a day, along with the optical density of the culture to know the growth of the pathogen.

![](https://static.igem.wiki/teams/4147/wiki/engineering/test-40b0a6.png)

**Test:**

A qualitative analysis of the data will be performed. And it will be determined whether siRNA has an effect on the pathogenicity of *P. capsici*.

**Learn:**

![](https://static.igem.wiki/teams/4147/wiki/engineering/learn-40b0a6.png)

In case we observe a positive effect we could perform a more reliable assay, such as real-time PCR. For this, the corresponding primers should be made to measure the effect of the siRNAs on the expression of the gene decided.

This type of assay will be able to confirm what we observed in the qualitative assay.

If necessary, the siRNAs should be redesigned.

**# **REFERENCES****

Cheng, W., Lin, M., Chu, M., Xiang, G., Guo, J., Jiang, Y., ... & He, S. (2022). RNAi-Based Gene Silencing of RXLR Effectors Protects Plants Against the Oomycete Pathogen Phytophthora capsici. Molecular Plant-Microbe Interactions, 35(6), 440-449.

Fakhr, E., Zare, F., & Teimoori-Toolabi, L. (2016). Precise and efficient siRNA design: a key point in competent gene silencing. Cancer gene therapy, 23(4), 73-82.

Horn, T., & Boutros, M. (2010). E-RNAi: a web application for the multi-species design of RNAi reagents—2010 update. Nucleic acids research, 38(suppl\_2), W332-W339.

Lawson, C. E., Harcombe, W. R., Hatzenpichler, R., Lindemann, S. R., Löffler, F. E., O'Malley, M. A., García Martín, H., Pfleger, B. F., Raskin, L., Venturelli, O. S., Weissbrodt, D. G., Noguera, D. R., & McMahon, K. D. (2019). Common principles and best practices for engineering microbiomes. Nature reviews. Microbiology, 17(12), 725–741. https://doi.org/10.1038/s41579-019-0255-9

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**#### Overview**

On this page you can check out the laboratory protocols we used for each one of the experiments we did.  **Click on each button to read the protocol.**

![ iRNAldo welcoming you to the experiments page](https://static.igem.wiki/teams/4147/wiki/education/irnaldos/l-experiments.png)

Competent Cells

[**Go to Results**

![](https://static.igem.wiki/teams/4147/wiki/bus.png)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

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{% extends "layout.html" %}
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[Achievements](#achievements)
[Biobrick Assembly](#assembly)
[First Round of Experimentation](#firstround)
[Second Round of Experimentation](#secondround)
[Third Round of Experimentation](#thirdround)
[Troubleshooting](#troubleshooting)
[Successes](#successes)
[Unsuccess](#unsuccess)
[Overall Conclusion](#CONCLUSION)
[References](#references)

![](https://static.igem.wiki/teams/4147/wiki/education/irnaldos/l-results.png)

**#### Overview**

The purpose of the experimental work was to express the DrsB1 and PcOSM constructs in the selected chassis *E. coli* BL21 (DE3), in order to produce these recombinant antimicrobial peptides with the objective of developing a fungicidal solution. This section describes the experimental work performed during the past months. An explanation of the assembly of the registered constructs and the strains we used for transformation to obtain the antimicrobial peptides of interest are shown. Additionally, the expression of mCherry (a monomeric RFP) was evaluated in *E. coli* BL21 (DE3) with a regulated LacI promoter and lambda pL promoter hybrid. Finally, we analyzed and interpreted both successful and unsuccessful results, where we were able to define the opportunity areas as well as to provide information on the troubleshooting that can help future iGEM teams.

**# **ACHIEVEMENTS****

The creation and registry of sixteen new BioBricks™, each containing the genetic code for the expression of the target proteins:

**Table 1.** Parts added to the registry.

| Sequence | Accession Number | Abbreviation | Status |
| --- | --- | --- | --- |
| ![PcOSM schematic representation](https://static.igem.wiki/teams/4147/wiki/parts/1pcosm.png) | [BBa\_K4147000](http://parts.igem.org/Part%3ABBa_K4147000) | N/A | Registered |
| ![(CBD)2-DrsB1 schematic representation](https://static.igem.wiki/teams/4147/wiki/parts/1drsb1.png) | [BBa\_K4147001](http://parts.igem.org/Part%3ABBa_K4147001) | N/A | Registered |
| ![Expression cassette schematic representation](https://static.igem.wiki/teams/4147/wiki/k4147001.png) | [BBa\_K4147002](http://parts.igem.org/Part%3ABBa_K4147002) | N/A | Transformed |
| ![Expression cassette schematic representation](https://static.igem.wiki/teams/4147/wiki/k4147003.png) | [BBa\_K4147003](http://parts.igem.org/Part%3ABBa_K4147003) | N/A | Transformed |
| ![Expression cassette schematic representation](https://static.igem.wiki/teams/4147/wiki/bba-k4147004-scheme.png) | [BBa\_K4147004](http://parts.igem.org/Part%3ABBa_K4147004) | DrsB1 | Transformed |
| ![Expression cassette schematic representation](https://static.igem.wiki/teams/4147/wiki/1dsba.png) | [BBa\_K4147006](http://parts.igem.org/Part%3ABBa_K4147006) | N/A | Registered |
| ![Expression cassette schematic representation](https://static.igem.wiki/teams/4147/wiki/k4147007.png) | [BBa\_K4147007](http://parts.igem.org/Part%3ABBa_K4147007) | PcOSM | Transformed |
| ![Expression cassette schematic representation](https://static.igem.wiki/teams/4147/wiki/k4147008.png) | [BBa\_K4147008](http://parts.igem.org/Part%3ABBa_K4147008) | DrsB1-improved | Transformed |
| ![Expression cassette schematic representation](https://static.igem.wiki/teams/4147/wiki/bba-k4147004-scheme.png) | [BBa\_K4147009](http://parts.igem.org/Part%3ABBa_K4147009) | N/A | Registered |
| ![Expression cassette schematic representation](https://static.igem.wiki/teams/4147/wiki/k4147010.png) | [BBa\_K4147010](http://parts.igem.org/Part%3ABBa_K4147010) | mCherry-LacI | Transformed |
| ![Expression cassette schematic representation](https://static.igem.wiki/teams/4147/wiki/k4147011.png) | [BBa\_K4147011](http://parts.igem.org/Part%3ABBa_K4147011) | mCherry-Hybrid | Transformed |
| ![Expression cassette schematic representation](https://static.igem.wiki/teams/4147/wiki/parts/up.png) | [BBa\_K4147012](http://parts.igem.org/Part%3ABBa_K4147012) | Up part of cassette for siRNA production of RXLR gene for P. capsici | Registered |
| ![Expression cassette schematic representation](https://static.igem.wiki/teams/4147/wiki/parts/down.png) | [BBa\_K4147013](http://parts.igem.org/Part%3ABBa_K4147013) | Down part of cassette for siRNA production | Registered |
| ![Expression cassette schematic representation](https://static.igem.wiki/teams/4147/wiki/parts/1reverse.png) | [BBa\_K4147014](http://parts.igem.org/Part%3ABBa_K4147014) | Reverse of BBa\_K525998 (RBS and promoter) | Registered |
| ![Expression cassette schematic representation](https://static.igem.wiki/teams/4147/wiki/parts/1treverse.png) | [BBa\_K4147015](http://parts.igem.org/Part%3ABBa_K4147015) | Reverse of BBa K864600 (Terminator) | Registered |
| ![Expression cassette schematic representation](https://static.igem.wiki/teams/4147/wiki/parts/1sirna.png) | [BBa\_K4147016](http://parts.igem.org/Part%3ABBa_K4147016) | RXLR siRNA | Registered |
\*DrsB1 is also known as MsrA2

**# **BIOBRICK ASSEMBLY****

The parts (DrsB1, PcOSM-LacI, DrsB1-improved, mCherry-LacI, mCherry-Hybrid promoter) were synthesized with the standard prefix and suffix flanking the regions of interest to facilitate restriction. The PcOSM, DrsB1 and DrsB1-improved parts were also synthesized by changing the LacI promoter to a lambda pL hybrid promoter (although these were not registered). Once the sequences were obtained, multiple cycles of PCR were performed with the objective of adding extra nucleotides on both ends to ensure a double digestion with the restriction enzymes EcoRI and PstI, to finally insert said sequences on the expression vectors ligating with MasterMix Ligase. Thanks to this, the complete expression constructs were obtained (Figure 1).

![BioBrick™ BBa_K4147008](https://static.igem.wiki/teams/4147/wiki/fig1-1-results.png)

[![ BioBrick™ BBa_K4147004](https://static.igem.wiki/teams/4147/wiki/figure2-results.png)](#podcastImpact)

![](https://static.igem.wiki/teams/4147/wiki/education/irnaldos/t-attributions.png)

![BioBrick™ BBa_K4147007](https://static.igem.wiki/teams/4147/wiki/fig1-3-results.png)

![BioBrick™ BBa_K4147010](https://static.igem.wiki/teams/4147/wiki/fig-1-4-results.png)

![BioBrick™ BBa_K4147011](https://static.igem.wiki/teams/4147/wiki/fig-1-5-results.png)

**Figure 1.** SnapGene® maps of A) BioBrick™ BBa\_K4147008, B) BioBrick™ BBa\_K4147004, and C) BioBrick™ BBa\_K4147007 ligated into backbone pSB1C3. D) BioBrick™ BBa\_K4147010. E) BioBrick™ BBa\_K4147011.

The last two constructs (mCherry-LacI (D) and mCherry-Hybrid (E)) were created with the purpose of analyzing the different promoters and comparing the results with the predictions made through the mathematical model.

Finally, the transformation of said constructs into *E. coli* BL21 (DE3) was carried out by means of heat shock. Clones were then selected for plasmid extraction and visualization by electrophoresis. All this was possible to achieve during the past months by carrying out the experimentation process described next, however the detailed protocols can be found in the  [experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) section.

**# **FIRST ROUND OF EXPERIMENTATION****

We amplified the sequences received from IDT® with the Taq Platinum polymerase at 50ºC and 55°C. After several PCR attempts, the PcOSM part with the hybrid promoter was obtained as expected (Figure 2). This allowed us to standardize the PCR protocol and add bases at both ends to later perform restriction digest.

![PcOSM with hybrid promoter amplification.](https://static.igem.wiki/teams/4147/wiki/figure-2-results-2.png)

The next step after standardization of the PCR protocol was to amplify the remaining sequences received from IDT. As seen in Figure 3, the sequences DrsB1-improved with both the LacI promoter and the hybrid promoter were amplified correctly at 50°C and 55°C. The PcOSM insert with a hybrid promoter was also amplified successfully at 60°C and 65°C, which meant we could proceed to ligate them into a backbone (this with a previous purification with the PureLink™ Quick Gel Extraction Kit to remove unspecified products). On the other hand, when trying to amplify PcOSM with the LacI promoter, no product appeared at either temperature.

![Comparison between amplifications of inserts with LacI promoter and hybrid promoter in a 0.8% agarose gel.](https://static.igem.wiki/teams/4147/wiki/fig3-results.png)

Simultaneously we tried to purify pJUMP28-1A for the further ligation of the parts, so we did a Miniprep extraction after transformation, but the expected band wasn’t obtained on the electrophoresis gel (Figure 3 well B). After many unsuccessful attempts to recover said plasmid, we ceased to keep trying at this idea.

We amplified the DrsB1 insert with the LacI promoter again at 50°C and 55°C and the expected band size of 540 bp was obtained (Figure 4). This confirmed that we had one of the constructs ready to purify with the PureLink™ Quick Gel Extraction Kit to remove unspecific bands for subsequent digestion and ligation with the expression vector.

![DrsB1 with LacI promoter amplification.](https://static.igem.wiki/teams/4147/wiki/figure-4-results-2.png)

At this point we had amplified the following sequences:

* PcOSM with a LacI promoter.
* PcOSM with a hybrid promoter.
* DrsB1-improved with a hybrid promoter.
* DrsB1-improved with a LacI promoter.
* DrsB1 with a LacI promoter (existing Part).

We decided to order the synthesis again with adapter sequences which would add base pairs at the ends allowing for direct digestion without amplification. Other than saving time, amplification errors would be avoided since the used polymerase wasn’t of high fidelity.

**# **SECOND ROUND OF EXPERIMENTATION****

Subsequently, digestion was carried out with EcoRI and PstI restriction enzymes. Initially, we performed the ligation in the pUC19 cloning vector in order to standardize the ligation protocol. The protocol was applied when ligating DrsB1-improved, DrsB1, and PcOSM into dephosphorylated pSB1C3 and later transforming into *E. coli* BL21(DE3).

Additionally, the inserts of mCherry-LacI, mCherry-Hybrid, (CBD)2-DrsB1, and PcOSM without DsbA with vector pTWIST Kan High Copy were also transformed into *E. coli* BL21(D3).

A colony PCR was made for the transformed constructs with the GoTaq Flexi DNA polymerase to confirm the presence of the insert, and the expected band sizes were obtained. The results were visualized in a 0.8% agarose gel as shown in Figure 5.

![Colony PCR from E. coli BL21 (DE3) transformants.](https://static.igem.wiki/teams/4147/wiki/figure-5-results-2.png)

We performed a Miniprep extraction to prove the presence of the DrsB1 in pSB1C3 plasmid with mCherry-LacI as the positive control.

![Miniprep extraction DrsB1 in pSB1C3](https://static.igem.wiki/teams/4147/wiki/figure-6-results-2.png)

After having successful ligation we confirmed that the constructs were correctly designed and had the potential to express. In order to confirm that, we advanced on to the third experimentation round.

**# **THIRD ROUND OF EXPERIMENTATION****

**## **Protein production****

Isopropyl β-D-1 thiogalactopyranoside (IPTG) was used to induce protein expression. This reagent activates the Lac promoter by preventing the Lac repressor from performing its repressor activity, since it cannot bind to the operator region. Extraction with B-PER ™ was then performed to extract intracellular proteins. This strategy resulted in problems expressing the antimicrobial peptides (DrsB1-improved, DrsB1 and PcOSM), therefore, we decided to start performing the mathematical model induction assays with mCherry-LacI and mCherry-Hybrid promoter.

**## **mCherry Induction****

The induction of both mCherry inserts (with a LacI promoter and with the lambda pL hybrid promoter) was done with constructs from Twist Bioscience, both expressing a 26.7 kDa protein. These were transformed into *E. coli* BL21 (DE3) by means of heat shock. As shown in Figure 7, an acceptable transformation efficiency was achieved. Growth in the culture medium alone would indicate a successful transformation; however, the color of the clones is another indicator that the plasmid insertion was successful.

![  E. coli BL21 (DE3) transformed with mCherry-LacI (left) and mCherry-Hybrid promoter(right).](https://static.igem.wiki/teams/4147/wiki/fig8-results-2.png)

We later performed induction using IPTG, growing transformed *E. coli* BL21 (DE3) for 4 hours and adding 0 mM, 0.25 mM, 0.50 mM, 0.75 mM and 1.0mM IPTG for another 4 hours. The culture was then diluted 3:1 with LB broth added with kanamycin in order to be loaded onto a 96-well plate and read. These samples were also analyzed with an SDS-PAGE.

For the induction of the mathematical model assays, an SDS-PAGE was performed using a different construct of mCherry of known molecular weight as a control. The expected protein had a weight of 31.1 kDa due to its 2 His-tags. The induction of mCherry-LacI and mCherry-Hybrid promoter was also performed.

As shown in Figure 8, lanes D-J have pronounced bands larger than 30 kDa, whereas lanes B-C have no such bands, implying that the mCherry protein was expressed. However, there are no discernible differences between hours of induction and IPTG concentration.

![Polyacrylamide gel 12% for visualization of the SDS-PAGE results of the mCherry construct induction with different IPTG concentrations and the whole induction kinetic.](https://static.igem.wiki/teams/4147/wiki/fig8-results.png)

The gel in Figure 9 shows the induction of the 31.1 kDa mCherry protein (lanes B-E) and the 26.7 kDa mCherry-Hybrid promoter protein (lanes F-J), with thick and defined bands in each case. Similarly, expression can be detected but no differences in concentrations or induction times.

![ Polyacrylamide gel 12% for visualization of the SDS-PAGE results of the mCherry control and mCherry-Hybrid promoter construct induction with different IPTG concentrations and the whole induction kinetic.](https://static.igem.wiki/teams/4147/wiki/fig9-results.png)

Furthermore another induction attempt (Figure 10) showed mCherry-LacI protein bands (Lane D-G) even in those samples not induced (D, F, H) while these were not found in the controls (B-C) nor in those wells loaded with the PcOSM sample (H-I).

![ Polyacrylamide gel 12% for visualization of the SDS-PAGE results of the E. coli BL21 induced and not, mCherry-Hybrid promoter induced and not, and also PcOSM without DsbA induced and not.](https://static.igem.wiki/teams/4147/wiki/fig10-results.png)

These results showed that there was protein expression even when no IPTG was added based on the similarities among the banding patterns at consecutive hours and concentrations, as well as its similarity with the control (*E. coli* BL21 (DE3)). Additionally, the strong pink color on figure 7 demonstrates protein production even after the induction protocols began.

The gels show unsuccessful obtainment of the desired protein. Since the results did not come out as expected we opted for looking for possible explanations that could help us understand the results and solutions.

**# **TROUBLESHOOTING****

After several induction attempts, we still got no results, so we decided to investigate further on the possible sources of error in the experiments. Firstly we analyzed the *E. coli* strain used. The BL21 (DE3) system contains the gene that codes for the T7 RNA polymerase under the control of an IPTG inducible promoter [(Hernández *et al*., 2021).](#R2) However the T7 polymerase is highly selective when heavily induced, producing up to 50% of the expected recombinant protein. Additionally, this strain is deficient in the Ion and ompT proteases, both in charge of degrading proteins during the purification process [(Quiroga, 2012).](#R4)

Besides the selected strain, the induction parameters were also reviewed, including the IPTG concentrations, where the kinetics of induction against time reflected the changes in concentration and the quality of the IPTG. Correspondingly, kinetics of up to 8 hours were set up, even though bibliography states that 4 hours is enough to consider a kinetic assay done. Likewise, 3 inductor concentrations (0.1 mM, 0.5 mM and 1.0 mM) were used on the constructs, using other concentrations to the assay for the mCherry constructs (0.25 mM, 0.50 mM, 0.75 mM, and 1.0 mM). On every induction experiment fresh IPTG would be prepared to achieve max quality.

Due to time constraints in the lab, we were not able to test all of our hypotheses. You can read more about this on the [Engineering](https://2022.igem.wiki/tec-chihuahua/engineering) page.

**# **Successes****

* The creation and registry of sixteen new BioBricks™.
* The assembly of six new composite BioBricks: PcOSM [[BBa\_K4147000]](http://parts.igem.org/Part%3ABBa_K4147000), PelB-(CBD)2-DrsB1 [[BBa\_K4147001]](http://parts.igem.org/Part%3ABBa_K4147001), Expression construct for (CBD)2-DrsB1 [[BBa\_K4147002]](http://parts.igem.org/Part%3ABBa_K4147002), PcOSM Construct with LacI regulated promoter [[BBa\_K4147003]](http://parts.igem.org/Part%3ABBa_K4147003), mCherry construct with LacI regulated promoter for bacterial expression [[BBa\_K4147010]](http://parts.igem.org/Part%3ABBa_K4147010), and mCherry construct with lacI regulated, lambda pL hybrid promoter for bacterial expression [[BBa\_K4147011]](http://parts.igem.org/Part%3ABBa_K4147011). All of them are expression systems for antimicrobial peptides.
* Characterization of LacI [[BBa\_R0010]](http://parts.igem.org/Part%3ABBa_R0010) and Lambda [[BBa\_R0011]](http://parts.igem.org/Part%3ABBa_R0011) promoters.

**# **Unsuccesses****

* After several experiments with the IDT synthetic sequences, we realized that the DNA was not in optimal conditions, thus soliciting a reposition of the product, and simultaneously ordering new sequences from Twist Bioscience to avoid time delays.
* At the time of amplifying the constructs with PCR we had problems given that we couldn't identify the correct alignment temperatures for the primers [[BBa\_G1004]](http://parts.igem.org/Part%3ABBa_G1004) and [[BBa\_G1005]](http://parts.igem.org/Part%3ABBa_G1005). Therefore, we did a temperature gradient of 50-65°C, identifying an optimal temperature of 55ºC where minimum unspecific products were obtained.
* For weeks, we had problems with the ligation protocol. Molar ratios of 3:1, 5:1, and 7:1 were tried with no success. Following that, mass ratios of 3:1, 5:1, and 7:1 were tested. We also tried different ligases such as T4 ligase from Invitrogen and Anza™ T4 DNA ligase Master Mix with different quantities of enzymes ranging from 1 to 5 µL at [5 units/µL]. Nevertheless, we realized that the best combination of parameters consisted of a 3:1 mass proportion, dephosphorylated vector and using the Anza™ T4 DNA ligase Master Mix.

**# **Overall Conclusion****

From the beginning we knew we would have setbacks, especially due to the nature of our ambitious goals and what we wanted to reach. Likewise, we are aware of the areas for improvement that we had during these eight months, as well as the future refinements that should be done experimentally in order to achieve the proposed solution. Finally, even though we did not obtain satisfactory results, these will be very useful for future iGEM teams, as well as helping us to continue analyzing the viability and scalability of our solution beyond the competition.

**# **REFERENCES****

González, A., & Fillat, M. F. (2018). *Aspectos metodológicos de la expresión de proteínas recombinantes en Escherichia coli*. Revista de educación Bioquímica, 37(1), 14-27.

Hernández-Alcántara, G., García-Torres, I., & Alba-Martínez, Z. (2021). *Expresión de proteínas recombinantes en un sistema heterólogo*

Kesik-Brodacka, M., Romanik, A., Mikiewicz-Sygula, D., Plucienniczak, G., & Plucienniczak, A. (2012). *A novel system for stable, high-level expression from the T7 promoter*. Microbial cell factories, 11, 109. https://doi.org/10.1186/1475-2859-11-109

Quiroga Campano, A. L. (2012). *Optimización del cultivo de Escherichia coli para la producción de cutinasas recombinantes*.

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