iGEM 2022 — Team rum-uprm

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* [Home](index)
* Team

  [Team](team)
  [Attributions](attributions)
  [Collaborations](collaborations)
* Project

  [Description](description)
  [Research Optimization](research-optimization)
  [Experiments](experiments)
  [Notebook](notebook)
  [Results](results)
  [Engineering Success](engineering)
  [Hardware](hardware)
  [Model](model)
  [Proposed Implementation](implementation)
  [Proof of concept](proof-of-concept)
  [Future works](future-works)
  [Contribution](contribution)
* Parts

  [Parts](parts)
* [Safety](safety)
* Human Practices

  [Integrated Human Practices](human-practices)
  [Education & Communication](communication)
* [Awards](education)

# Notebook

In this page you will find wet lab procedures performed in the laboratory.

![](https://static.igem.wiki/teams/4460/wiki/img/illustrations/rum-igem/component-vector.png)
![](https://static.igem.wiki/teams/4460/wiki/img/illustrations/rum-igem/component-vector-2.png)

* [Overview](#overview)
* [Monthly Progress](#section1)
* [Laboratory Notebook](#section2)

## Overview

---

We started working on the logistics, research, and sequence optimization and design from January 2022 to June 2022. Weekly meetings were held to monitor the Biology Team progress. Alongside, every month a set of tasks were assigned to each team member regarding the variety of challenges presented. Additional meetings with our PI's, advisor and other collaborators were done to assess the route of our project. Laboratory work began in June 2022 starting with a week of workshops regarding the basic techniques required for our cloning plan. Modifications to the plan were carried out each month forward until we arrived at our final cloning plan. Due to the effects of Hurricane Fiona our laboratory work had to pause for two weeks. Unfortunately, the lack of electrical service caused a loss in samples and laboratory materials that needed to be kept at optimal temperatures. We hope to eventually continue our lab work and obtain the desired results of our cloning plan to move forward with our proof of concept.

## Monthly Progress

---

##### 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.

![Biology Research Symposium](https://static.igem.wiki/teams/4460/wiki/biology-research-symposium.jpg)

**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.

![Biology Lab Workshop](https://static.igem.wiki/teams/4460/wiki/biology-team-workshops.jpg)

**Figure 2.** Biology team members taking laboratory workshop.

## Laboratory Notebook

---

To organize our laboratory plan, work-flow, protocols, and assistance records we used Benchling, a digital laboratory notebook. The notebook was divided by weeks of work, however, we compiled them into months as presented below. The [Biology Team](https://2022.igem.wiki/rum-uprm/team) was working simultaneously on both devices in which the cloning plans were modified a total of three times. See our [Results](https://2022.igem.wiki/rum-uprm/results) and [Engineering Success](https://2022.igem.wiki/rum-uprm/engineering) for more details of each redesign. Each biology team member that participated in the wetlab was able to perform each protocol presented at least once to promote a learning experience within the team.

![LBB Preparation](https://static.igem.wiki/teams/4460/wiki/img-0521.jpg)

**Figure 3.** Preparation of Luria Bertani Broth (LBB).

[June 2022](https://static.igem.wiki/teams/4460/wiki/june-2022.pdf)
[July 2022](https://static.igem.wiki/teams/4460/wiki/july-2022-compressed.pdf)
[August 2022](https://static.igem.wiki/teams/4460/wiki/august-2022-compressed.pdf)
[September 2022](https://static.igem.wiki/teams/4460/wiki/september-2022-compressed.pdf)

© 2022 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at [gitlab.igem.org/2022/rum-uprm](https://gitlab.igem.org/2022/rum-uprm).

[![logo](https://static.igem.wiki/teams/4460/wiki/photos-igem2022/logo-igem-blanco-wiki.png)](index)

* [Home](index)
* Team

  [Team](team)
  [Attributions](attributions)
  [Collaborations](collaborations)
* Project

  [Description](description)
  [Research Optimization](research-optimization)
  [Experiments](experiments)
  [Notebook](notebook)
  [Results](results)
  [Engineering Success](engineering)
  [Hardware](hardware)
  [Model](model)
  [Proposed Implementation](implementation)
  [Proof of concept](proof-of-concept)
  [Future works](future-works)
  [Contribution](contribution)
* Parts

  [Parts](parts)
* [Safety](safety)
* Human Practices

  [Integrated Human Practices](human-practices)
  [Education & Communication](communication)
* [Awards](education)

# Engineering Success

In this page you will find details about our project design this year.

![](https://static.igem.wiki/teams/4460/wiki/img/illustrations/rum-igem/component-vector.png)
![](https://static.igem.wiki/teams/4460/wiki/img/illustrations/rum-igem/component-vector-2.png)

* [Overview](#overview)
* [Research](#section1)
* [Design](#section2)
* [Build](#section3)
* [Test](#section4)
* [Learn](#section5)
* [References](#section6)

## Overview

---

Synthetic biology involves the engineering and redesigning of biological elements; therefore, it is important to follow the cycle of design, build, test, and learn (DBTL) to obtain a successful project. Although we initially planned to complete the cloning and sequencing of our three devices, a flaw was noted in the promoter of our detection circuit since all of the homologous regulators were not found in E. coli. This prompted us to re-design the module for future implementation and develop another circuit for activation of our degradation device. This design would allow us to build and test the latter module. The cloning plan consisted of the bacterial transformation of 10-beta competent cells to make a double insertion of DNA fragments using an adaptation of 3A assembly and simple restriction enzyme cloning respective to each device. As some challenges emerged, the plans changed to single insertion of DNA fragments and a simple restriction enzyme cloning for the bacterial transformation in ec100. After three cycles of DBTL, we were able to design a promising circuit. Due to unforeseen meteorological circumstances, we were not able to continue building and testing our current design. However, after many trials of re-designing, we achieved the theoretical proposal of a circuit that is now ready for implementation, thus helping us move one step closer to our final goal.

## Research

---

Our goal as a team is to develop a modern solution to a problem that for decades has been a concern to the citizens of Vieques. This municipal island of Puerto Rico, was the site of a military bombing range from the 1940s to 2003. This factor has created a local and national predicament, due to the contamination issues created by the bomb detonations that occurred near its several bodies of water (Whitall et al., 2016). For example, Anones Lagoon, is cataloged as one of the most contaminated in Vieques.

According to Whitall et. al, the contamination in Vieques can be a risk to human health. Due to the large amount of contaminants, the seafood safety guidelines are being affected. Moreover, a test performed in Casa Pueblo in 2013 revealed that the marine productivity around Vieques had dropped 90% over the last 10 years. For such reasons, the team decided to find a possible solution to this problem with the use of synthetic biology.

The waters of Vieques have a variety of types of contaminants. As a team, we wanted to focus on hexahydro-1,3,5-trinitro-s-triazine (RDX). RDX was chosen because the exposure to this explosive is known to be extremely dangerous. According to the agency of toxicity in Atlanta, RDX can affect blood pressure on humans and be carcinogenic if there is a large amount of exposure.

## Design

---

As a team we noticed that some of the homologous regulators in Algd promoter, on Device 1, weren’t found on E. coli. Due to the fact that the purpose of this device is the expression of luxl and luxR, to generate AHL, the primary promoter activator of the RDX degradation device; we decided to search for other promoter option for Device 1. All of these with the goal of completing the RDX degradation device proof of concept.

The supplementary Device 1, begins with the PLac- Luxl composite. The transcription is induced by Isopropyl ß-D-1-thiogalactopyranoside, which leads to the expression of the Luxl gene. This gene helps catalyze the conversion of S-adenosylmethionine (SAM) into acyl-homoserine lactone (AHL), which is able to diffuse across cell membranes and the AHL molecules are recognized by LuxR receptors. Then the device has an RFP protein generator which serves as a biological marker that helps us confirm the first part of the device.

The LuxR is regulated by the PTet promoter, so it can constitutively express.
Our main goal with the original Device 1 was to detect RDX and with it, begin the transcription and expression of genes to convert SAM to AHL and activate the inducible promoter of Device 2. For this reason the team is searching for a solution. One of them can be the hmp/hcp fusion promoter.

## Build

---

We designed cloning workflows for our devices using BioRender allowing us to visualize where each restriction enzyme would cut and how the fragments would be assembled to form our desired product. We also used Benchling to facilitate the view of our genetic circuits. Initially, we started using 10-beta competent E. coli cells which were later changed to EC100 competent cells. When attempting to build or clone our genetic circuits some difficulties were presented. For each cloning plan, explained in detail in Experiments, modifications were made to become closer to our built devices. Elements such as restriction enzyme and ligase functionality, vector usage, and procedure calculations led us to verify the correct implementation of each procedure in the cloning process. Assessment of the protocols led us to conclude that for our final assembly we should use a new ligase and ligase buffer. In addition, insertion of one fragment at a time in the vector would also help us catalog potential problems in the ligation procedure since less variables were involved. Due to Hurricane Fiona our team was not able to finish building the devices. However, every new building strategy helped us get closer to our goal by identifying possible defects in our cloning plans.

## Test

---

We designed our genetic circuits in a way that could be monitored and only function when certain conditions were met. For that reason, each device incorporates inducible promoters that can only be activated in a desired environment and contain reporters that allow us to track the transcription of each device. The testing portion of our project consisted in performing gel electrophoresis and verifying our transformed cells. When we did not obtain the desired results in each protocol the team verified the probable causes and fixed the building process. These iterations would eventually lead us to the final assembly of our device. Since the cloning of the devices was not possible during this cycle the other testing elements regarding the reporter genes and elements from our proof of concept were not performed to completion.

## Learn

---

As we designed, built and tested our devices, we encountered setbacks that allowed us to discover faults in our designs. Primarily, we learned that the AlgD promotor of our Device 1 needed homologous regulators that were not all found in E. coli. Therefore, upon further research, we decided that the most reasonable solution was to conduct a promoter fusion of the promoters of the hmp and the hcp E. coli genes, which together are responsible for the detection of RDX. On another note, we also encountered problems with our protocols, cloning plans and calculation errors, from which we learned and corrected in order to see progress in our results. Moreover, while using iGEM’s linearized plasmids, pSB1A3 and pSB1K3, we were unable to obtain a ligated construct and decided to use pSB1C3 instead. With each cycle of DBTL, we fixed each mistake we encountered and were able to optimize our prototypes. Unfortunately, after designing a promising circuit, the building of this design was not able to be fulfilled because of a meteorological phenomenon that hit our island and left it without power.

## References

---

Whitall, D.R., A.L. Mason, M. Fulton, E. Wirth, D. Wehner, A. Ramos-Alvarez, A.S. Pait, B. West, E. Pisarski, B. Shaddrix, and L. Reed. 2016. Contaminants in Marine Resources of Vieques, Puerto Rico. NOAA Technical Memorandum NOS NCCOS 223. Silver Spring, MD. 70 pp. http://dx.doi.org/10.7289/V5/TM-NOS-NCCOS-223

Envioramental Protecction Agency. 2014.Technical fact sheet hexahydro-1,3,5-trinitro-s-triazine (RDX). Technical Fact Sheet – Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) (epa.gov)

© 2022 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at [gitlab.igem.org/2022/rum-uprm](https://gitlab.igem.org/2022/rum-uprm).

[![logo](https://static.igem.wiki/teams/4460/wiki/photos-igem2022/logo-igem-blanco-wiki.png)](index)

* [Home](index)
* Team

  [Team](team)
  [Attributions](attributions)
  [Collaborations](collaborations)
* Project

  [Description](description)
  [Research Optimization](research-optimization)
  [Experiments](experiments)
  [Notebook](notebook)
  [Results](results)
  [Engineering Success](engineering)
  [Hardware](hardware)
  [Model](model)
  [Proposed Implementation](implementation)
  [Proof of concept](proof-of-concept)
  [Future works](future-works)
  [Contribution](contribution)
* Parts

  [Parts](parts)
* [Safety](safety)
* Human Practices

  [Integrated Human Practices](human-practices)
  [Education & Communication](communication)
* [Awards](education)

# Experiments

In this page you will find the respective workflows of our different wet lab procedures.

![](https://static.igem.wiki/teams/4460/wiki/img/illustrations/rum-igem/component-vector.png)
![](https://static.igem.wiki/teams/4460/wiki/img/illustrations/rum-igem/component-vector-2.png)

* [Overview](#overview)
* [Previous Cloning Plan](#section1)
* [Final Cloning Plan](#section2)
* [Workflow](#section3)
* [Protocols](#section4)
* [References](#section5)

## Overview

---

During this cycle, our team performed a diversity of experiments to achieve our objectives. The iGEM-RUM cloning plan was divided into two devices, with Device 1: AHL and luxR generator using DNA from the iGEM DNA Kit Plates and Device 2: Biodegradation of RDX, using DNA designed by iGEM-RUM 2021 and subsequently ordered from Integrated DNA Technologies (IDT). Cloning for these parts into our EC100™ E. coli competent cells was done by following a simple restriction endonuclease digestion protocol for both devices in which they were digested using the prefixes and suffixes provided to us by iGEM. Digestion of our individual inserts was followed by ligation and transformation into our pSB1C3 vector. From here, subsequent ligation of both devices into one same cell was intended so that the cell could be functional for AHL and LuxR generation and RDX biodegradation.The final cloning plan for our Device 1 and Device 2 followed the workflow shown below with their respective restriction sites and restriction endonucleases as seen in Figures 1 and 2.

## Previous Cloning Plan

---

##### **Device 1**

During our time working in the lab, our cloning plan was optimized twice, for a total of three cloning strategies. However, due to an unforeseen meteorological event which compromised our samples, we were unable to finalize the last cloning strategy. Our initial cloning strategy for Device 1 (the supplementary version) consisted of digesting iGEM’s linearized plasmid, pSB1A3, with EcoRI-HF and PstI-HF. D1P1 [(BBa\_K876014)](http://parts.igem.org/Part%3ABBa_K876014) was digested with EcoRI and SpeI while D1P2 [(BBa\_K081014)](http://parts.igem.org/Part%3ABBa_K081014) was digested with XbaI and PstI-HF. The resulting ligation would be digested with SpeI-HF and PstI-HF for the insertion of D1P3 [(BBa\_K116640)](http://parts.igem.org/Part%3ABBa_K116640) digested with XbaI and PstI-HF.

For our second cloning strategy, we utilized the digested pSB1C3 plasmid from the iGEM kit plate parts (D1P2 and D1P3), extracted and purified from agarose gel. Since we no longer used the pSB1A3 linearized backbone, we opted for a simpler insertion method. D1P1 would serve as the vector for D1P2 and D1P3 inserts. In order to linearize the plasmid while keeping the D1P1 insert, we digested D1P1 with SpeI-HF. Then, D1P2 was digested with XbaI and SpeI-HF producing a downstream insertion after ligation (A1). The A1 construct was then linearized with SpeI-HF for the insertion of D1P3. Like D1P2, D1P3 was digested with XbaI and SpeI-HF.

##### **Device 2**

Similar to Device 1, we employed the use of restriction cloning as our cloning strategy for Device 2. The genetic circuit for this device was designed in Benchling and divided into two fragments; both were ordered through Integrated DNA Technologies. These were delivered as linearized fragments which contain iGEM’s restriction site prefix and suffix. The cloning strategy for Device 2 changed twice. The initial cloning strategy for Device 2 consisted of using EcoRI-HF and SpeI-HF enzyme master mix alongside a XbaI and PstI-HF enzyme master mix for Device 2 Fragment 1 (D2F1) and Device 2 Fragment 2 (D2F2), respectively. Then, iGEM’s linearized vector, pSB1K3, was digested with a EcoRI-HF and PstI-HF enzyme master mix. After successful ligation, the result would be a circularized plasmid with D2F1 located upstream from D2F2.

[First Cloning Plan](https://static.igem.wiki/teams/4460/wiki/cloning-plans-july-3-2022.pdf%20)

## Final Cloning Plan

---

Our final cloning plan for Device 1 consists of simple restriction endonuclease digestion. This device contains a pSB1C3 vector, the Plac-luxI composite (D1P1) [BBa\_K876014](http://parts.igem.org/Part%3ABBa_K876014), the RFP protein generator (D1P2) [BBa\_K081014](http://parts.igem.org/Part%3ABBa_K081014), and the LuxR pTet regulated (D1P3) [BBa\_K116640](http://parts.igem.org/Part%3ABBa_K116640). To assemble Device 1 we digested D1P1 at the SpeI-HF and PstI-HF restriction sites and D1P2 with XbaI and PstI-HF. The idea behind selecting these sites is because for D1P1, we wanted to maintain the part and backbone attached (D1P1-BB). However, for D1P2 we needed to detach the part from its backbone. This site also ensured D1P2 was inserted downstream in D1P1-BB. After inactivating the digestion reactions, D1P1-BB and D1P2 were ligated for five hours in the thermocycler to secure a consistent temperature. The product of this ligation was named A1. Since the restriction sites (iGEM prefix and suffix) are conserved after ligation, the same steps performed for A1 were then repeated for A2: the insertion of the third part (D1P3) into the vector containing the first and second parts. This would complete our cloned Device 1.

![](https://static.igem.wiki/teams/4460/wiki/device-1.png)

**Figure 1.** Supplementary Device 1 Workflow

For Device 2, the final restriction endonuclease cloning plan was done by using our first fragment of the second device, D2F1, and ligating it to our vector backbone psB1C3. This backbone was extracted from D1P1 to ensure functionality of the vector. This first assembly was named B1. This procedure was done using the enzymes XbaI and SpeI-HF to digest our vector and insert D2F1. After this, a second digestion took place to insert the second fragment, D2F2, into B1. For this, we used the endonucleases SpeI-HF and PstI-HF to linearize B1 and used XbaI and PstI-HF to cut D2F2. Once we ligated our parts into a single vector the second assembly, B2, was generated culminating our Device 2.

![](https://static.igem.wiki/teams/4460/wiki/device-2-1.png)

**Figure 2.** Device 2 Workflow

## Workflow

---

Both of our devices followed a similar cloning procedure. The traditional cloning by restriction endonuclease digest that was performed in the laboratory to insert our Device 1 into the EC100™ E. coli competent cells used the following general protocol:

Resuspension (Fig. 3) was conducted with the iGEM DNA Kit Plates retrieved from the iGEM Distribution Kits using the parts 22M and 6C by utilizing the corresponding wells to acquire the genes of interest following the protocol established by iGEM in 2019. The genes of interest were subsequently transformed into competent EC100™ E. coli cells by electroporation and left to grow overnight.

![](https://static.igem.wiki/teams/4460/wiki/workflow-device-1-final.png)

**Figure 3.** Workflow for Device 1

This was followed by restriction endonuclease digestion, which was performed utilizing type II restriction endonucleases EcoRI and XbaI prefixes and SpeI and PstI suffixes (Fig. 3) following the protocol from New England Biolabs.

To confirm these parts had been successfully cloned into the competent E. coli cells and digested correctly by the restriction endonucleases, gel extraction and purification was performed following the Monarch Gel Extraction Kit (Fig. 4) by excising DNA samples directly from the low melting point agarose gel, followed by purification of the gel to be ligated as one genetic circuit.

![](https://static.igem.wiki/teams/4460/wiki/dna-agarose-gel-extraction-and-purification.png)

**Figure 4.** DNA Agarose Gel Extraction and Purification

After confirming all of the digested parts that were to be assembled eventually into a pSB1C3 vector, ligation was performed using the Cohesive End Ligation Protocol by New England Biolabs utilizing the Hi-T4 DNA Ligase (Fig. 1). Once ligation was performed, transformation of the competent cells was executed again with the assembled parts (Fig. 5) to complete the genetic circuit for Device 1 using the method mentioned previously for transformation of competent E. coli cells.

![](https://static.igem.wiki/teams/4460/wiki/transformation.png)

**Figure 5.**Transformation Protocol

The results from the transformation were plated into petri dishes (Fig. 3) prepared with the corresponding Chloramphenicol antibiotic as a selective measure and left overnight until colony growth was observed. Subsequently, the colonies that grew were inoculated into liquid medium using Luria Bertani Broth (Fig. 3) and left to grow overnight. This DNA underwent Qiagen Mini Prep to extract the plasmid DNA. Later on, this extracted DNA was put in gel electrophoresis to confirm that our DNA was successfully assembled.

The assembly of our second device, for biodegradation, followed a similar procedure (Fig. 6).

![](https://static.igem.wiki/teams/4460/wiki/workflow-device-2-final.png)

**Figure 6.** Workflow for Device 2

The general protocol differed in the primary steps of cloning, since our synthesized fragments for the Device 2 genetic circuit were ordered from Integrated DNA Technologies. Unlike the first device that came in a plasmid, the device 2 inserts came already linearized and thus needed to be digested before being ligated to a plasmid. Digestion of the inserts was done following the restriction endonuclease digestion procedure mentioned previously, according to their weight and calculations. This procedure was followed by agarose gel electrophoresis for confirmation of our individual parts and later on, gel extraction and purification to eventually ligate all of our individual parts into our plasmid of choice, pSB1C3. Once this procedure was done for both devices we would have proceeded to join both devices in a single chassis (Fig. 7).

![](https://static.igem.wiki/teams/4460/wiki/final-double-plasmid-insertion-2.png)

**Figure 7.** Final Double Plasmid Insertion

## Protocols

---

[Miniprep](https://static.igem.wiki/teams/4460/wiki/qiaprep-spin-miniprep-protocol.pdf)
[Agarose Gel Electrophoresis](https://static.igem.wiki/teams/4460/wiki/agarose-gel-electrophoresis-protocol.pdf)
[Double Endonuclease Digestion Reaction](https://static.igem.wiki/teams/4460/wiki/double-digestion-protocol.pdf)
[Gel Extraction](https://static.igem.wiki/teams/4460/wiki/monarch-dna-agarose-gel-purification-protocol.pdf)
[DNA Ligation Reaction](https://static.igem.wiki/teams/4460/wiki/dna-ligation-reaction-protocol.pdf)
[Bacterial Transformation](https://static.igem.wiki/teams/4460/wiki/bacterial-transformation-protocol.pdf)
[Inoculation of Transformed Cells](https://static.igem.wiki/teams/4460/wiki/inoculation-of-transformed-cells-protocol.pdf)

## References

---

Darmstadt, I. T. U. (2022). https://static.igem.org/mediawiki/2020/1/1f/t--tu\_darmstadt--methods\_tu\_darmstadt\_2020.pdf. In iGEM TU Darmstadt 2020 Methodic Protocols and Materials, Antibiotic Preparation. https://static.igem.org/mediawiki/2020/1/1f/T--TU\_Darmstadt--Methods\_TU\_Darmstadt\_2020.pdf

Fixed Cell Pellet. (n.d.). UMass Chan. https://www.umassmed.edu/morphology/protocols/fixed-cell-pellet/

Frozen Stock Culture. (n.d.). HarveyLab. https://haveylab.horticulture.wisc.edu/wp-content/uploads/sites/66/2016/05/Preparation-of-frozen-bacterial-stock-cultures.pdf

Biolabs, N. E. (n.d.). Monarch® DNA gel extraction kit protocol (NEB #T1020). NEB. https://international.neb.com/protocols/2015/11/23/monarch-dna-gel-extraction-kit-protocol-t1020

GoldBio TE Buffer Stock Solution. (n.d.). GoldBio. https://www.goldbio.com/documents/3590/TE%20Buffer%20Stock%20Solution-10X.pdf

IGEM DNA Kit Plate Resuspension. (2019). IGEM. http://parts.igem.org/Help:2019\_DNA\_Distribution

iGEM-RUM. (n.d.). Prepare antibiotic-supplemented Luria-Bertani (LB) Agar plates SOP. https://static.igem.org/mediawiki/2021/b/b6/T--RUM-UPRM--Device23ACloning.pdf

Optimizing Restriction Endonuclease Reactions. (n.d.). New England Biolabs. https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions

Protocol for Dephosphorylation of 5´-ends of DNA using CIP (NEB #M0290). (n.d.). New England Biolabs. https://international.neb.com/protocols/0001/01/01/protocol-for-dephosphorylating-with-cip

Protocol for Hi-T4TM DNA Ligase (NEB #M2622). (n.d.). New England Biolabs. https://international.neb.com/protocols/2019/10/10/protocol-for-hi-t4-dna-ligase-neb-m266

QIAGEN. (2020). Miniprep and Transformation Validation.

TA cloning method: Fragment Resuspension. (n.d.). Integrated DNA Technologies. https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/protocol/gblocks-fragment-t-a-cloning-protocol.pdf?sfvrsn=e41c3407\_8

Thawing Frozen Stock Culture. (n.d.). ATCC. https://haveylab.horticulture.wisc.edu/wp-content/uploads/sites/66/2016/05/Preparation-of-frozen-bacterial-stock-cultures.pdf

© 2022 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at [gitlab.igem.org/2022/rum-uprm](https://gitlab.igem.org/2022/rum-uprm).

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  [Description](description)
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# Results

In this page you will find details about the project's results and challenges.

![](https://static.igem.wiki/teams/4460/wiki/img/illustrations/rum-igem/component-vector.png)
![](https://static.igem.wiki/teams/4460/wiki/img/illustrations/rum-igem/component-vector-2.png)

* [Challenges](#overview)
* [Project Achievements](#section1)

## Challenges

---

##### **AlgD promoter**

When optimizing the AlgD promoter, it was observed that our promoter's sequence did not match the one synthesized. It was also discovered that the AlgD promoter lacked any regulators, including the sigma 22 regulator, which serves as the recognition site for transcription to begin. This implies that this promoter would never have been active.

##### **pSB1A3 and pSB1K3**

While using iGEM’s linearized plasmids, pSB1A3 and pSB1K3, we were unable to obtain a ligated construct resulting in the redesigning of our cloning plan. Upon failure, we observed that the pSB1A3 plasmid did not generate the predicted EcoRI/PstI digest, and that pSB1A3 and pSB1K3 were incompatible with various RFC. We also predicted that because of the various freeze-thaw cycles it had endured since it had been acquired by the previous cycle, the DNA could have been degraded by nucleases and is therefore no longer viable for use. As a result, we modified our cloning technique for Devices 1 and 2, opting to use the pSB1C3 vector for every assembly.

##### **Overhang compatibility**

While performing the second cloning strategy for Device 1, we quickly realized that digesting the backbone (D1P1) with only one enzyme could cause multiple mistakes when ligating. Not only could the vector quickly recircularize since it has compatible overhangs, the inserts could be ligated backwards since both insert overhangs are compatible with the SpeI restriction site. On account of this realization, we changed our cloning strategy for Device 1 by digesting D1P1-BB with two enzymes, SpeI and PstI.

##### **Inoperative T4 DNA Ligase and T4 DNA Ligase Buffer**

Throughout our time in the laboratory, we were able to obtain definitive results for various protocols including miniprep, digestion reactions, agarose gel purification, bacterial transformation, among others. However, we were unable to successfully ligate any constructs. We presumed that the T4 DNA Ligase Buffer was not working efficiently due to lack of ATP, caused by multiple freeze-thaw cycles. Unfortunately, while we were ligating the A1 construct (D1P1-BB and D1P2) with a new T4 DNA Ligase and T4 DNA Ligase Buffer, we coincided with the arrival of the devastating Hurricane Fiona which left our lab without power for two weeks, leaving many materials compromised and our project without conclusive results.

![](https://static.igem.wiki/teams/4460/wiki/positive-and-negative-controls-of-d1p1-bb-ligation.png)

**Figure 1.** Unsuccessful ligation of D1P1-BB [SpeI-HF] positive control (09/13/2022). In order to test if the Hi-T4 DNA Ligase we were using was working, two controls were performed. These were plated on LBA supplemented with chloramphenicol. The positive control consisted of D1P1-BB linearized with SpeI-HF then ligated with NEB’s Hi-T4 DNA Ligase. On the other hand, the negative control included D1P1-BB linearized with SpeI-HF but in the ligation reaction, Hi-T4 DNA Ligase was substituted for Nuclease free water. Absence of bacterial growth was expected on the negative control, yet not on the positive control. Lack of colony growth after 24 hours on the positive control was indicative of inefficient DNA Ligase activity.

## Project Achievements

---

##### Device 1: AHL and LuxR protein Generator

*Successful results*

* Confirmation of D1P1, D1P2 and D1P3 parts.
* Successful plasmid purification, digestion, gel purification and transformation protocols.

![](https://static.igem.wiki/teams/4460/wiki/d1p2-grown-on-plate.png)

**Figure 2.** D1P2 grown colonies on LBA plate supplemented with chloramphenicol (08/09/2022). EC100™ cells were electroporated with the digested D1P2 [XP] product. The plate was observed after 24 hours of incubation at 37ºC. Once colonies were inoculated in LBB and the QIAgen miniprep was completed, agarose gel electrophoresis was performed as seen in Figure 3.

![](https://static.igem.wiki/teams/4460/wiki/d1p2-confirmation.jpg)

**Figure 3.** Successful D1P2 [XbaI and PstI-HF] digestion (08/11/2022). Digestion products were separated by electrophoresis on 1% agarose and compared to NEB’s 1 kb+ DNA ladder in the first lane. Expected weights confirm digestion of D1P2, observed in the third lane. D1P2 C2 was the second colony inoculated in LBB from the plate shown in Figure 2.

![](https://static.igem.wiki/teams/4460/wiki/gel-electrophoresis.png)

**Figure 4.** Successful digestion of D1P1 [SpeI-HF and PstI-HF] and D1P3 [XbaI and PstI-HF] (09/08/2022). Digestion products were separated by electrophoresis on 1% agarose and compared to NEB’s 1 kb+ DNA ladder in the first lane. All lanes were consistent with their expected insert and vector weights. For the second and third lane, D1P1 [SP] was located around the 3,000 bp line since the plasmid was linearized and did not lose the insert. Lanes 3, 4 and 5, D1P3 [XP], showed weights nearing 2070 bp for the pSB1C3 vector and 999 bp for the D1P3 insert.

*Unsuccessful results*

* Ineffective ligation of A1.

##### Device 2: RDX Biodegradation

*Successful results*

* Successful plasmid purification, digestion, gel purification and transformation protocols.

*Unsuccessful results*

* Ineffective ligation of B1.

![](https://static.igem.wiki/teams/4460/wiki/transformation-of-d2f1-psb1k3.png)

**Figure 5.** Unsuccessful D2F1 + pSB1K3 ligation attempt on LBA plate supplemented with kanamycin (08/19/2022). EC100™ cells were electroporated with the ligation product. After 96 hours of incubation at 37ºC, colony growth was not observed.

![](https://static.igem.wiki/teams/4460/wiki/b1.jpeg)

**Figure 6.** Unsuccessful B1 ligation (D2F1 + pSB1C3) attempt on LBA plate supplemented with chloramphenicol (09/09/2022). EC100™ cells were electroporated with the B1 ligation product. The plate was observed after 24 hours of incubation at 37ºC. Upon examination, we realized that the second B1 ligation attempt was unsuccessful and a contaminant was present.

© 2022 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at [gitlab.igem.org/2022/rum-uprm](https://gitlab.igem.org/2022/rum-uprm).