Difference between revisions of "Team:RUM-UPRM/Attributions"
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<p style="margin-left:7%; margin-right:7%">I would like to recognize the hard work of the iGEM RUM-UPRM team. Our expectations for this iGEM cycle were very different; due to the pandemic, we had to alter our plans, adapt to virtual meetings, and accept we were not going to have access to the laboratory, a very important part of the development of an iGEM project. Although there were many difficulties, the team pulled through and persistent to create this amazing project. We look forward to the further development of Mer-Nite to the Rescue so it can have a positive impact in Vieques and could have further applications. </p> | <p style="margin-left:7%; margin-right:7%">I would like to recognize the hard work of the iGEM RUM-UPRM team. Our expectations for this iGEM cycle were very different; due to the pandemic, we had to alter our plans, adapt to virtual meetings, and accept we were not going to have access to the laboratory, a very important part of the development of an iGEM project. Although there were many difficulties, the team pulled through and persistent to create this amazing project. We look forward to the further development of Mer-Nite to the Rescue so it can have a positive impact in Vieques and could have further applications. </p> | ||
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<div id="section3" style="background-color:rgba(255,0,0,0.1)"> | <div id="section3" style="background-color:rgba(255,0,0,0.1)"> | ||
<h1>RDX Genetic Circuit</h1> | <h1>RDX Genetic Circuit</h1> | ||
Revision as of 05:58, 26 October 2020
Attributions and Acknowledgements
Attributions
I would like to recognize the hard work of the iGEM RUM-UPRM team. Our expectations for this iGEM cycle were very different; due to the pandemic, we had to alter our plans, adapt to virtual meetings, and accept we were not going to have access to the laboratory, a very important part of the development of an iGEM project. Although there were many difficulties, the team pulled through and persistent to create this amazing project. We look forward to the further development of Mer-Nite to the Rescue so it can have a positive impact in Vieques and could have further applications.
The Biology Team, composed of Patricia, Luis G., Elimar, Rigo, Melissa, Elmer, and Elan were responsible for the design and cloning model of the genetic circuits. Patricia, the team leader, also wrote the safety plan for the precautions in the lab due to COVID-19.
The Engineering Team, composed of Mariela, Natalia, and Claudia, were responsible for the design of mathematical models applied to the genetic circuits. They also helped in the determination of the parameters for the Bioreactor Design for Synthetic Biology Applications.
The Human Practices Team, composed of Gabriela, Ana Sofía, and Emily, were involved in contacting stakeholders and setting up virtual meetings with them. They also organized the 2nd edition of the Synthetic Biology Week of the University of Puerto Rico in Mayagüez, held virtually this year due to the pandemic. With the additional help of Claudia, the Human Practices Team also organized the SynBio 101: Summer Camp for High School Students. Other members of the team served as mentors to the high school students and helped deliver the materials to the students to different parts of the Island.
A group of us were in charge of the administrative work. Andrea was responsible for the finances and fundraising; Elan redacted and answered emails and wrote many letters; Marieli was in charge of the maintenance of our social media accounts, the design of promotional material, and created the COVID-19 precautions brochure; Natalia communicated with the many offices of our University; and Claudia did a little bit of everything and was the leader in the team meetings. Natalia and Marieli also were in charge of the collaborations with other teams. Luis M. organized the Bioreactor Design for Synthetic Biology Applications and was in charge of the logistics and Paula was in charge of communicating with the three teams and organizing the project.
Our artistic team members designed the various creative outlets that are part of our project. Elmer, with the help of Claudia, assembled and designed the promo video. Elmer also drew the project logo and created the presentation video with the help of Claudia and Elan. Elimar drew the postcard for a collaboration and designed various biological diagrams. Emily designed the team spirit poster for the World Meetup, the Vieques information brochure, a figure for the promo video, and the logo for the SynBio101: Summer Camp. Elmer also created the presentation video with the help of Claudia and Elan.
Carlos was responsible for the design of the wiki.
RDX Genetic Circuit
The RDX device is composed of three genetic circuits, each with a specific function: detection, biodegradation, and lysis, which will be regulated/controlled by quorum sensing. This device begins with the stress sensitive promoter algD, which will initiate transcription in the presence of RDX. Later, LuxI gene will create a synthase capable of creating acyl-homoserine lactones (AHL) that will bind to LuxR protein. The binding of these two molecules creates a transcription factor that will activate LuxpR promoter. It will then begin the transcription of the second device, that will contain the xplAB gene, which produces enzymes capable of degrading RDX. After transcription of xplAB gene has been completed, the GFP gene will be transcribed. GFP allows us to identify whether xplAB enzymes are being produced by emitting a green fluorescence. The end-products of RDX, specifically nitrite, and formaldehyde, will act as transcription factors in the third device, which is the killswitch. Lastly, the kill switch circuit will be controlled by a modified synthetic-AND Gate, which will allow bacterial lysis by requiring the presence of metabolized products such as Formaldehyde and Nitrate to maximize biodegradation of RDX. Lysis will initiate due to the presence of colicin and, therefore, stop bacterial transcription.
Figure 1: ......
Device #1: Detection of RDX
In the presence of RDX, algD promoter will initiate the transcription. As the transcription begins, luxI gene will convert S-adenosil metionina (SAM) into acyl-homoserine lactone (AHL); consequently the luxR will produce a protein which binds to AHL. This merge will stimulate the transcription of luxpr (pLuxR) promoter in the second device.
Figure 2: ......
| Part | Function |
|---|---|
| algDpromoter | Transcription of this promoter begins due to a stress response towards RDX. |
| LuxI BBa_C0061 | This synthase converts SAM into a small molecule called an acyl-homoserine lactone (AHL), which can diffuse across cell membranes. |
| LuxR BBa_C0062 | When bound to AHL, it produces a protein that can stimulate transcription from the right hand lux promoter (LuxpR). |
Device #2: Biodegradation of RDX
The LuxpR promoter will be upregulated by the activation of LuxR activator protein, which forms a complex with autoinducer AHL. As a result, the xplAB system will catalyze the reductive denitration and subsequent ring cleavage of RDX. When biodegradation of RDX is complete the gene GFP, a green fluorescent protein, will function as a reporter gene.
Figure 3: ......
| Part | Function |
|---|---|
| luxpR BBa_R0062 | Promoter that will be up-regulated by the activation of LuxR activator protein which forms a complex with autoinducer AHL. This promoter is the key element to produce proteins of interest, increase the rate of transcription, and mediate the final effects of quorum-sensing. |
| xplA and xplB genes | Involved in the catalyzation of the reductive denitration and ring cleavage biodegradation pathways for the organic contaminant RDX. The xplB gene encodes for a partner flavodoxin reductase, while the xplA encodes for flavodoxin domain fused (at the N-terminus) of a P450 cytochrome. |
| merB BBa_K1420002 | An organomercurial lyase that cleaves the binding between organic radicals and mercury, releasing Hg(II). |
| GFP with degradation LVA tag BBa_K592010 | Involved in the expression of green fluorescence protein, as well, encodes for a small peptide functioning as a degradation tag that will allow for fine-tuning protein levels and thus regulating of the GFP in the bacteria. |
Device #3: Killswitch of RDX Circuit
To maximize efficiency of our prototype, we decided to harbor the use of modified synthetic-AND Gate as our killswitch, which was originally developed by Christopher A. Voigt and modified by the Peking University 2009 iGEM team. Our synthetic AND Gate requires the use of two inputs, formaldehyde and nitrite, which are the byproducts of biodegraded RDX, to generate the protein colicin, which causes cellular lysis. The lysis gene (output) will have an inducible T7 promoter which will be activated with the corresponding T7 RNA polymerase. This polymerase (T7ptag) will be added to the circuit and will be regulated by an inducible promoter, PyeaR, which will activate in the presence of nitrite. However, this polymerase will have two amber mutations, which are nonsense mutations that inhibit the complete translation of the polymerase. To overcome the nonsense mutation, a tRNA amber mutation suppressor, SupD, will be controlled by a formaldehyde-inducible promoter, Pfrm. This means that lysis will only occur if both nitrite and formaldehyde are present.
Figure 4: ......
| Part | Function |
|---|---|
| Pyear promoter BBa_K216005 | Inducible promoter that will be activated in the presence of nitrate, nitric oxide, or nitrite. |
| Formaldehyde-Inducible Promoter BBa_K2728001 | Inducible promoter that will be activated in the presence of formaldehyde. |
| SupD + terminator BBa_K228100 | A tRNA coding gene and it can be well terminated by the terminator BBa_B0015. |
| T7ptag (T7polymerase with amber mutation) BBa_K228000 | A coding sequence of T7 polymerase with two Amber mutations. The transcription of T7ptag gene can only lead to the generation of its mRNA, further translation into T7 RNA polymerase is blocked because of the amber mutation. |
| PT7 BBa_K2406020 | When the T7 RNAP is present it permits levels of transcription. |
| Lysis gene BBa_K117000 | This gene encodes for the lysis protein in colicin-producing strains of bacteria that will result in an interruption of the system by lysis of the bacteria. |
References
Add references.