Notebook

January 2022

Our team decided to stay with the same project as the last cycle’s team: R-DetoX. iGEM-RUM 2022 decided to work on optimizing our project, aiming on working in proof of concepts of our devices and cloning our first and second device. In addition, from January 12 to January 27, each team member from Biology Team, Social Impact Team, and Engineering Team was assigned to complete the CITI program certificates for the basic course on Social & Behavioral Research.

February 2022

On February 1st, we had our first meeting with Luis A. García Cruz, and he introduced our Biology Team members into the various cloning techniques. We compared the different methods and started thinking about which one would be the best option for our project. On February 4, 5, and 6, intensive workshops were held where the entire team was educated on safety measures, aseptic techniques, preparation of media, reagents and antibiotics, synthetic biology, the basis of our project, techniques and basic laboratory protocols, use of Benchling and presentations about each team division (biology, engineering, and social impact). At the beginning and ending of the intensive workshops a pre-test and post-test was offered to evaluate the learning progress of our team members. On February 20, the overall objectives of the biology team were established. The objectives included: propose primers for Gibson Assembly use, choose cloning plan method, make a proposal for the water sampling, make proposals for sponsorships, practice laboratory techniques, begin laboratory work, verify sequences of each device, reach out to collaborations with the island of Vieques, review proof of concept, and create efficient deadlines for each deliverable. To review the laboratory technique experience level of the team a questionnaire was offered. Additionally, during the meeting the team was divided into three sub-teams where each created a presentation explaining the devices (1, 2 and 3) developed during the last cycle to the other team members. Device 1 was presented on February 28 by Arianna C. López Pérez and Enoelis Viera Ortiz. At the beginning of March Device 2 was presented by Lyan E. Lugo Collado and Valeria A. Ramirez Rivera, while Device 3 was presented by Shante Perez Nieto and Lina Blanco Rodríguez.

March 2022

During the first weeks of March the three sub-teams presented the three devices to the other team members. Additionally, a meeting was held to verify the genetic sequences with Luis García. The team members Arianna C. López Pérez and Shante Perez Nieto were present in this meeting. On March 10 the team was reassigned into three groups to research and collect information about: terminal adapters, primers, and establishing communication with IDT. The team members responsible for working on the terminal adapters were Arianna C. López Pérez, Lyan E. Lugo Collado, and Airinés González Velázquez. The team members responsible for working on the primers for Gibson Assembly were Shante Perez Nieto, Lina Blanco Rodríguez, and Debora D. Bermonthy Carlo. Lastly the communication with IDT was established by Keysha N. Resto Bravo, Aleysha Vargas Carrero, and Enoelis Viera Ortiz. The last weeks of March were spent doing research of all three categories (terminal adapters, primers, and establishing communication with IDT) and doing meetings about how to optimize the fragments.

April 2022

Luis Montalvo González verified the sequences and a progress report was generated for Victor Lopez Rivera to review. On April 12 a meeting was held to talk about the cloning method, non-coding sequences, AlgD operon and Luxpr promoter. Additionally, tasks were assigned to research: the regulatory genes of the AlgD operon, variants of the Luxpr promoter where it only activates under the correct stimulus, and vector options. In regards to the sequences, non-coding sequences were added to the beginning and end, respectively, of every fragment to facilitate ligation. Another important event of this meeting was the decision to select 3A assembly as our preferred cloning method for the project. On April 20 the optimizations were discussed with our PI at the time, Dr. Carlos Ríos Velázquez. During this meeting additional tasks were assigned to verify the non-coding sequences and restriction enzyme sites, verify vector options, characterization of the algD promoter and the LuxR gene, redact the materials and methods for the cloning method selected. The tasks were divided evenly among all the biology team members. On April 27 the biology team reassured that 3A assembly and traditional cloning would be the only cloning methods to be used. During the vector research pET vectors were considered. Since last cycles spacer was part of a pET vector and the NdI enzyme is common among those vectors, tasks were assigned to review if there would be conflict. Once the modified sequences were completed, codon optimization and a Blast was performed. Additionally, inventory was held to see what materials were available in the lab for our wet lab phase.

May 2022

On May 4 a meeting was held with Luis García Cruz to discuss various questions generated while researching. One concern was the complexity of the fragments which was accepted-moderate due to the added non-coding sequences. A few suggestions were to add different non-coding sequences to push the other inwards, to put two different ones, or to change a non-coding sequence with lower GC content. For the vector selection it was discussed that perhaps in an experimental sense a low copy vector would not be viable. Additionally, for 3A assembly a simple vector like pUC19 would suffice. For the remainder of May the research above mentioned continued to prepare everything for laboratory implementation. During this month the fragments for device 2 were ordered. Planification of workshops were begun for them to be held during the month of June. Pre-labs and supplementary materials were given so each team member could have prior knowledge of all the techniques to be practiced. Every biology team member that wanted to participate in the lab had to complete a test made by Dr. Carlos Ríos Velázquez that covered safety protocols, basic laboratory techniques and the identification of the materials found in each part of the lab. The members who completed this test and therefore participated in the wet lab were: Aleysha Vargas Carrero, Lina Blanco Rodríguez, Lyan E. Lugo Collado, Débora D. Bermonthy Carlo, Keysha N. Resto Bravo, Arianna C. López Pérez, Airinés González Velázquez, Shante Perez Nieto, Amanda C. Rivera Morales, Luis Montalvo Rivera, and our president Solimar Muñiz Acevedo. In addition, on May 18 we participated in the Biology Research Symposium of our university biology department under the category of Biotechnology.

Figure 1. iGEM-RUM presenting at the Biology Research Symposium at Univeristy of Puerto Rico-Mayagüez.

June 2022

During June 1 a meeting was held to clarify doubts about the workshops and the necessary information to know. To evaluate the learning progress a Kahoot was performed. On June 8 a meeting was held with our PI at the time Dr. Carlos Ríos Velázquez were he recommended that our proof of concept be done on E. coli since Pseudomonas naturally degrade RDX in nature, to research literature regarding the homologous regulators and sigma factor in E. coli of the regulators and sigma factor responsible for the AlgD promoter, and verify which regulators were truly essential for the promoter functionality. In the middle of June laboratory protocols began as we attended workshops offered by our advisor Víctor Lopez while simultaneously working on research to make a decision to stay with or eliminate the AlgD promoter.