Engineering Success

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

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)

The repository used to create this website is available at gitlab.igem.org/2022/rum-uprm.