Here you will find information about our project, the thought process behind it and our inspiration.
The Road to Coli. CTRL: SynBio101
SynBio 101
Our first step was to educate and inform the Academic community outside and inside our University. To achieve this, we started raising awareness of the synthetic biology field by offering info-tables and info-sessions. Simultaneously, we became pioneers by participating in the first Synthetic Biology University course in Puerto Rico. We then extended this initiative Island-wide by organizing the first ever Synthetic Biology Summer Camp; where fifty students from public and private High Schools were able to learn about Synthetic Biology and Recombinant DNA concepts. We further promoted the learning experience by asking each group to gather the knowledge obtained throughout the camp to create their own original prototypes and present these to their friends and families during the camp's closing ceremony.
To continue our mission, this semester we offered intensive Genetic Engineering and Recombinant DNA workshops. Thirty-five High School students were taught basic laboratory techniques such as Digestion, Ligation, Transformation and Gel Electrophoresis. The goal is that they can take these skills and knowledge and spread awareness in their schools, around the Island, and find synthetic biology solutions to the problems we face today.
October 15 2019, marked the beginning of the first Synthetic Biology Week in the University of Puerto Rico, Mayagüez Campus, and Puerto Rico. The first activity was an official Proclamation by our chancellor that recognized the SynBio week in our University. On Thursday, we had our main event. This was the first Synthetic Biology Carribean Talk where a conversation about this emerging field was started; 150 High School students and around 80 University students assisted. Synthetic biology, bioethics, graduate student projects, synthetic biology solutions, and genetic circuits were some of the topics discussed. The next activity was a Town Hall-type conversatory about regulations in the field and economic opportunities for Puerto Rico. Local politicians and the University community participated in this dialogue. The closing event involved School teachers, were teaching tools and possible course guidelines were provided.
All these events encompass our objective of informing, raising awareness, and educating the Academic, Professional and General public. Establishing the foundations of Synthetic Biology in Puerto Rico. The next steps would be to continue expanding our outreach and using our original prototype as a teaching tool.
Coli.CTRL
Before explaining what Coli.CTRL is, we want to give you a brief overview of some key concepts in order to facilitate the understanding of what our system does and how it works.
Genetic Material and Gene Expression
There are two types of genetic material: ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). For the purposes of this project we will be focusing on the latter. DNA is the hereditary material molecule of all living organisms and some viruses. It contains and transmits all the genetic information that makes up an organism. This information is stored as a “code” consisting of four different nucleotides: adenine, guanine, cytosine and thymine. The genome is the sum of all the organism’s DNA. Genes are short sections of DNA (group of three nucleotides aka codons) that serve as the instructions that will tell the cell what to do with the information stored in the DNA in order to make a functional molecule. Some genes code for protein molecules. These in turn are responsible for performing many functions inside a cell (the basic unit of life). Finally, the conversion of instructions into a functional “output” is referred to as gene expression.
Two Component Systems and Optogenetics
Two-component regulatory systems, commonly found in bacteria, are a type of mechanisms that allows the host organism to sense, respond and adapt to changing environmental conditions. These signaling systems consist of a membrane-bound histidine kinase that senses a specific stimulus and will transmit it to a partner response regulator that will determine the cellular response to this stimulus. This process is achieved via phosphorelay. This mediation of responses within the cells in order to help them adapt to changes in their environment include modification of protein interactions, catalysis of reactions and the initiation of the expression of target genes.
Optogenetics is a biological technique to control cells in living tissue using light and genetic engineering. Through the latter, cells are genetically modified to express light-sensitive ion channels. The end result of the technique will be cells that have been genetically modified in to order to express light-activates opsin proteins (light-sensitive transmembrane proteins).
Now you may be wondering how all of this information ties in with our project. Like we previously mentioned, bacteria naturally respond to different stimuli in their environment with the use of an innate signal pathway known as Two Component Systems (TCS); a sensor protein or better known as histidine kinase (HK)-response regulator (RR) circuit. Through a mechanism of phosphorylation between these proteins, a message is delivered and thus the activation of genes. Recent studies have demonstrated that if modified, this sensor protein can respond to light, giving it an optogenetic property. In this project we have decided to create a controllable, customizable and optimized gene expression circuit using this optogenetic approach as our stimulus. Here we expand on previous studies by generating a plasmid with all the building blocks necessary to become a predictable light-responsive gene expression system that can be expressed in Escherichia coli. Using Cph1, a light sensing domain, from Cyanobacteria Synechocystis and NarX-NarL, from Escherichia coli as our TCS of choice, as our fundamental building blocks. In addition, we incorporated multiple cloning sites (MCS) in strategic places to make this circuit customizable. We expect that by applying a specific wavelength to a re-engineered natural sensor from a TCS, gene expression will be induced resulting in the controllable and predictable production of a determined byproduct.
How does it work?
First, a Cph1-NarX fusion protein device that can transform a light stimulus to a chemical
signal which will start the transcription of the genes of interest. This bio-part is a combination of
a photoreceptor domain from a cyanobacteria, Synechocystis sp., fused by a linker to a histidine
kinase (HK) from Escherichia coli, NarX-NarL two-component system (TCS). The TCS are
composed of a sensor protein and a response regulator protein that are naturally found in
microorganisms. When the sensor protein senses the presence of a molecule, change of
temperature or ion, it sends a message by phosphorylation to its corresponding response regulator
which regulates gene expression. NarX-NarL, naturally responds to high concentrations of nitrite
and nitrate. Our modifications will allow this TCS to respond to a certain frequency of red light.
With our light-responsive fusion protein we can successfully activate gene expression.
Second, a strategically placed multiple cloning site (MCS) in a reporter gene positioned
after a NarL controlled promoter. The reporter gene that contains the MCS will be the green
fluorescent protein (GFP) gene, which also serves as a visual identifier for verifying the correct
insertion of the genes of interest. Resulting in a controlled expression device where the user can
interchange genes of interest that should be expressed upon light induced NarL regulation.
Tests will be conducted to further optimize the expression of genes and to determine the
optimum operating conditions for the system.
What is the output?
Throughout the course of the project’s development, there were several application ideas for the system. Deciding on a specific byproduct proved to be one of the most difficult tasks because after thorough research there were numerous propositions and uses that would, in theory, work out and produce a beneficial and innovative solution to current issues. This was because we were seeing the broad spectrum of applicable “outputs” for the project as a limitation rather than an advantage. After this realization we couldn’t help but ask: “If our device can be used for the consummation of so many different applications, why limit it to a single one?”. Instead of focusing on an output, we directed our efforts to the characterization of a controllable and customizable gene expression device that could be used for the synthesis of different byproducts. This means that our gene expression circuit can be used by researchers to assist their determined experiments. Sometimes in synthetic biology a project leads to new designs that carry out a specific function. Other times, hard work and discoveries are used to help future projects accomplish their goals, specifically ethically driven studies that are beneficial to the public. At iGEM RUM we believe both of these scenarios are important and beneficial to current and future generations. Determined to help “synthesize a better world” as our slogan says, we present to you our expression device: Coli.CTRL.
Inspiration behind our project
As students of the University of Puerto Rico, Mayagüez campus, we noticed that there is a vacuum for new fields in our institution. Because of this we decided to focus on promoting education and awareness of the synthetic biology field. We sought to impact the High School community so that in order to provide them with opportunities that we did not have. Our hope is that they can use these skills and develop their own projects to further science and find answers to the challenges we face today. With this purpsoe, we were motivated to create the SynBio 101 section of our project.
Coli.CTRL came next. From the beginning of our research, we focused on the control of gene expression from different stimuli. We were searching for the stimuli when we came across the paper titled Re-engineering a two-component system as light-regulated in Escherichia coli. We realized that we can use different light wavelengths to stimulate gene expression. We then realized that this prototype can be used to attend various problems that the Puerto Rican community face like the lack of new teaching tools, agricultural issues, and resolutions for the ever growing industries in the Island.
