iGEM 2019 — Team CCA_San_Diego

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# Team:CCA San Diego/Notebook

[Home](https://2019.igem.org/Team%3ACCA_San_Diego)

Team

[Team Members](https://2019.igem.org/Team%3ACCA_San_Diego/Team)
[Collaborations](https://2019.igem.org/Team%3ACCA_San_Diego/Collaborations)

Project

[Description](https://2019.igem.org/Team%3ACCA_San_Diego/Description)
[Design](https://2019.igem.org/Team%3ACCA_San_Diego/Design)
[Experiments](https://2019.igem.org/Team%3ACCA_San_Diego/Experiments)
[Notebook](https://2019.igem.org/Team%3ACCA_San_Diego/Notebook)
[Contribution](https://2019.igem.org/Team%3ACCA_San_Diego/Contribution)
[Results](https://2019.igem.org/Team%3ACCA_San_Diego/Results)
[Attributions](https://2019.igem.org/Team%3ACCA_San_Diego/Attributions)

Parts

[Parts Overview](https://2019.igem.org/Team%3ACCA_San_Diego/Parts)
[Basic Parts](https://2019.igem.org/Team%3ACCA_San_Diego/Basic_Part)
[Part Collection](https://2019.igem.org/Team%3ACCA_San_Diego/Part_Collection)

[Safety](https://2019.igem.org/Team%3ACCA_San_Diego/Safety)

Human Practices

[Human Practices](https://2019.igem.org/Team%3ACCA_San_Diego/Human_Practices)
[Education and Engagement](https://2019.igem.org/Team%3ACCA_San_Diego/Public_Engagement)

Awards

[Medal Criteria](https://2019.igem.org/Team%3ACCA_San_Diego/Medal_Criteria)
[Entrepreneurship](https://2019.igem.org/Team%3ACCA_San_Diego/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3ACCA_San_Diego/Hardware)
[Measurement](https://2019.igem.org/Team%3ACCA_San_Diego/Measurement)
[Model](https://2019.igem.org/Team%3ACCA_San_Diego/Model)
[Software](https://2019.igem.org/Team%3ACCA_San_Diego/Software)

[Judging](https://igem.org/2019_Judging_Form?team=CCA_San_Diego)

## Notebook

## Week 1 (June 3 - June 9)

* Started planning our iGEM summer camp at CCA and making presentations for interactive camp lessons
* Meetings regarding curriculum to be covered during the camp and what supplies we required
* Started our team’s wiki - added Project Description and a picture for the homepage

## Week 2 (June 10 - June 16)

* Wrapped up our presentations for the summer camp
* Gathered necessary supplies and materials to carry out day-to-day activities at camp
* Finalized our budget plans and registration for the camp

## Week 3 (June 17 - June 23)

* Hosted the CCA iGEM Summer camp with 15+ elementary and middle school students
* Taught techniques such as micropipetting, gel electrophoresis, and chromatography
* Exposed students to the basics of synthetic biology, nanotechnology, and biochemistry through interactive presentations
* Talked to and gained advice from Anthony Kang, a synthetic biology major at Northwestern University. Anthony helped us determine how to best construct our degradation pathways as well as some more advanced lab protocols.

## Week 4 (June 24 - June 30)

* Started communications with local labs and companies to find a lab space where we could perform our lab work
* Reviewed key documentation from last year’s project, and drafted out a lab schedule as well as a to-do list for things needed to be done

## Week 5 (July 1 - July 7)

* Compiled a lot of useful documents into a Literature Collective; the documents covered everything from the previous degradation of key PAHs (such as naphthalene and Chrysene) to approaches for biohydrogen synthesis
* Finalized our focus PAHs - Naphthalene, Chrysene, Fluorene, and Phenanthrene, while incorporating Salicylate and Phthalate as intermediate PAHs in these pathways
* Discussed ways to design constructs for the aforementioned PAHs’ degradation pathways

## Week 6 (July 8 - July 14)

* Started work on the PAH degradation constructs - focused on Chrysene and Naphthalene this week
* Discussed ways of theoretically testing constructs using MATLAB before actually ordering them (to determine any flaws we may miss)
* Expanded the Literature Collective to include more information about our target PAHs as well as biohydrogen production and synthesis

## Week 7 (July 15 - July 21)

* Reviewed last year’s Jamboree presentation to make sure we had all previous aspects in check* Started work on the PAH degradation constructs - focused on Phenanthrene and Fluorene this week

## Week 8 (July 22 - July 28)

* Received responses back from the J. Craig Venter Institute (JCVI) about potentially being able to use some of their lab space to carry out lab work; started a dialogue with them regarding the same* Wrapped up initial versions of our constructs (of degradation pathways for each of the 6 PAHs) - focused on Salicylate and Phthalate this week* Troubleshooted and attempted to fix mistakes in the PAH constructs

## Week 9 (July 29 - August 4)

* Continued our dialogue with JCVI scientists - came into contact with Dr. Frank Harrison and Dr. Michael Checkai* Started designing and researching protocol for our lab work - looked into Golden Gate Assembly and T4 ligation

## Week 10 (August 5 - August 11)

* Continued dialogue with JCVI - obtained permission for a lab tour and a basic-level discussion with JCVI employees* Continued designing and researching protocols - looked into BioBricks and compared its methods to Golden Gate Assembly

## Week 11 (August 12 - August 18)

* Looked for potential collaborations with other teams; made a spreadsheet that ranked compatibility with other teams based on project and proximity* Finalized setup with JCVI, asked for (and got) an opportunity to briefly discuss our projects with JCVI experts and have a detailed lab tour

## Week 12 (August 19 - August 25)

* Continued researching methods for protocol - looked into Gibson primers (overhangs) and bacterial transformation to test our plasmids* Attended the JCVI lab tour as well as had a discussion with Dr. Harrison and Dr. Checkai.

## Week 13 (August 26 - September 1)

* Finalized constructs for target PAHs (phenanthrene, fluorine, chrysene, and naphthalene)* Completed safety training prior to starting JCVI lab work - the safety training included Chemical and Biological Safety Standards

## Week 14 (September 2 - September 8)

* Finalized constructs for HSU-ETCD, alkane, alkene, and alkyne* Ordered HSU-ETCD, alk1, alky2, nap2, newfl3, fl2, and newphe3 from Twist Bioscience* Had an initial meetup with TPHS and DNHS to discuss general aspects of each of our projects and talk about surveys of the general public regarding the commonalities of our projects.

## Week 15 (September 9 - September 15)

* Switched protocol from using Golden Gate Assembly to Gibson Assembly and workflow due to the restrictions of T4 ligations and the accessibility of overhangs in Gibson primers* Began modeling with MATLAB'S symbology tool - used individual units and values to theoretically model Chrysene and Naphthalene* Began lab work at JCVI - altered our constructs and started basic plasmid insertions* Discussed potential for Bioreactor to stimulate a controlled environment for the E. Coli* Started a Literature review based on our aforementioned literature collective - this week, we focused on Chrysene and documenting key past experiments related to Chrysene degradation and uses

## Week 16 (September 16 - September 22)

* Participated in the I Love a Clean San Diego Beach Cleanup - collected over 20 pounds of trash and got a chance to interact with our target communities* Interviewed 33 beachgoers on their views of bacteria, oil spills, and our project - gained key insights into public perception of our methods as well as common concerns* Continued modeling of different PAH degradation pathways - this week, we used individual units and values to theoretically model Phenanthrene and Fluorene* Looked further into pursuing a bioreactor after raised public concerns about the potential side effects of E. Coli in the environment - contacted environmental scientists and researchers at the University of San Diego to learn more about the process* Continued the literature review based on our aforementioned literature collective - this week, we focused on Naphthalene and documenting key past experiments related to Naphthalene degradation and uses* Continued JCVI lab work\*

## Week 17 (September 23 - September 29)

* Finalized all 4 PAH models on MATLAB and theoretically simulated them* Changed protocol of Gibson Assembly using Gibson primers and workflow.* Contacted GoMRI (Gulf of Mexico Research Initiative) scientists after being referred to them by the University of San Diego professors; talked to them about new bioreactor plans* Continued to work on IGEM wiki - started on the Human Practices pages* Continued the literature review based on our aforementioned literature collective - this week, we focused on Phenanthrene and documenting key past experiments related to Phenanthrene degradation and uses* Continued JCVI lab work\*

## Week 18 (September 30 - October 6)

* Collaborated with the University of Washington and EVRY France iGEM Teams to translate their synthetic biology curriculum into Spanish and Portugese* Received responses from GoMRI scientists - encouraged the idea of the bioreactor and provided some valuable insights which we incorporated into the overall design of our project* Hosted a meetup with the TPHS and DNHS iGEM Teams, where we gave practice IGEM presentations and other teams gave feedback on the content of the presentation and how it was given.* Continued the literature review based on our aforementioned literature collective - this week, we focused on Fluorene and documenting key past experiments related to Fluorene degradation and uses* 3D-modeled designs for a bioreactor* Continued to work on IGEM wiki - added rough drafts for the Parts, Hardware, and Modeling sections* Continued JCVI lab work\*

## Week 19 (October 7 - October 13)

* Wrote up Software code with C++ / Arduino to allow for both aerobic and anaerobic respiration (for the E. Coli to carry out both PAH degradation and biohydrogen synthesis)* Finished up the literature review based on our aforementioned literature collective - this week, we focused on Biohydrogen Synthesis and finalized all previous drafts for each PAHs review* Wrote up documents and applied for a Provisional Patent for our bioreactor’s design to further our team’s entrepreneurial pursuits* Continued to work on IGEM wiki - added initial drafts for the Entrepreneurship, Software, Measurement, Parts Overview, and Project Design pages* Continued JCVI lab work\*

## Week 20 (October 14 - October 20)

* Finished up Software code with C++ / Arduino to allow for both aerobic and anaerobic respiration (for the E. Coli to carry out both PAH degradation and biohydrogen synthesis)* Wrote up documents and applied for a Provisional Patent for our bioreactor’s design to further our team’s entrepreneurial pursuits* Continued to work on IGEM wiki - finalized all drafts of aforementioned pages as well as added a Medal Criteria page and finished up all other Wiki pages (like Basic Parts and Project Results)* Continued and finalized JCVI lab work\*

**\* - for more details on the lab work conducted at JCVI, please see the  [Experiments](https://2019.igem.org/Team%3ACCA_San_Diego/Experiments) page of our wiki!**


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# Team:CCA San Diego/Experiments

[Home](https://2019.igem.org/Team%3ACCA_San_Diego)

Team

[Team Members](https://2019.igem.org/Team%3ACCA_San_Diego/Team)
[Collaborations](https://2019.igem.org/Team%3ACCA_San_Diego/Collaborations)

Project

[Description](https://2019.igem.org/Team%3ACCA_San_Diego/Description)
[Design](https://2019.igem.org/Team%3ACCA_San_Diego/Design)
[Experiments](https://2019.igem.org/Team%3ACCA_San_Diego/Experiments)
[Notebook](https://2019.igem.org/Team%3ACCA_San_Diego/Notebook)
[Contribution](https://2019.igem.org/Team%3ACCA_San_Diego/Contribution)
[Results](https://2019.igem.org/Team%3ACCA_San_Diego/Results)
[Attributions](https://2019.igem.org/Team%3ACCA_San_Diego/Attributions)

Parts

[Parts Overview](https://2019.igem.org/Team%3ACCA_San_Diego/Parts)
[Basic Parts](https://2019.igem.org/Team%3ACCA_San_Diego/Basic_Part)
[Part Collection](https://2019.igem.org/Team%3ACCA_San_Diego/Part_Collection)

[Safety](https://2019.igem.org/Team%3ACCA_San_Diego/Safety)

Human Practices

[Human Practices](https://2019.igem.org/Team%3ACCA_San_Diego/Human_Practices)
[Education and Engagement](https://2019.igem.org/Team%3ACCA_San_Diego/Public_Engagement)

Awards

[Medal Criteria](https://2019.igem.org/Team%3ACCA_San_Diego/Medal_Criteria)
[Entrepreneurship](https://2019.igem.org/Team%3ACCA_San_Diego/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3ACCA_San_Diego/Hardware)
[Measurement](https://2019.igem.org/Team%3ACCA_San_Diego/Measurement)
[Model](https://2019.igem.org/Team%3ACCA_San_Diego/Model)
[Software](https://2019.igem.org/Team%3ACCA_San_Diego/Software)

[Judging](https://igem.org/2019_Judging_Form?team=CCA_San_Diego)

# Experiments

A description of the research, experiments, and protocols we used in our iGEM project.

All characterization and measurement data for our parts is on the corresponding Registry part pages.

## Project Development

Horizon 2.0, a continuation of last year's Horizon project, focused on the degradation of toxic compounds of oil spills known as PAHs (Polycyclic Aromatic Hydrocarbons), while striving to produce clean biohydrogen energy as a result. Horizon 2.0 officially kicked off in February 2019. First, our team conducted an extensive literature review to deepen our understandings of PAHs, biohydrogen energy, and plasmid assembly techniques such as Golden Gate and Gibson (see [Project Design](https://2019.igem.org/Team%3ACCA_San_Diego/Design)). We also investigated ways of testing several aspects of our project, such as the MATLAB modeling of our pathways as well as using H2 Blue and a scanning fluorometer for various measurements (see [Measurement](https://2019.igem.org/Team%3ACCA_San_Diego/Measurement)).

Our next order of business was to design our constructs and degradation pathways. With the aid of websites such as Uniprot, we were able to successfully construct these, and also test them theoretically using MATLAB's Symbiology tool (see [Modeling](https://2019.igem.org/Team%3ACCA_San_Diego/Model)). We were extremely fortunate to be able to receive lab space and invaluable guidance from the J. Craig Venter Institute (JCVI), a renowned bioinformatics research center here in San Diego. The main parts of our lab were conducted in July-October, ranging from the tweaking of the aforementioned constructs to the Gibson assembly of plasmids and subsequent bacterial transformations and observations.

One key aspect of our project's development was the idea of using a bioreactor. Inspired by scientists and public concerns about the potentially harmful impacts of E. Coli in the oceanic ecosystems, we successfully 3D modeled and designed a bioreactor. The integration of the bioreactor helped provide a safe and controlled environment for PAH degradation, thus eliminating concerns from our target communities about side-effects. The bioreactor also gave us the ability to simulate both aerobic and anaerobic respiration with our E. Coli, allowing the the simultaneous processes of PAH degradation and biohydrogen production. This switch between respiration types was also coded by our team using C++ and Arduino (see [Software](https://2019.igem.org/Team%3ACCA_San_Diego/Software)). Our team has also applied for a patent on the aforementioned bioreactor (see [Entrepreneurship](https://2019.igem.org/Team%3ACCA_San_Diego/Entrepreneurship)).

Overall, due to the large-scale negative impacts of oil spills such as the Deepwater Horizon Oil Spill and a local one near our community, our iGEM team was inspired to create Horizon 2.0 - a project aiming to degrade toxic compounds in PAHs while producing biohydrogen as a by-product.

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:CCA San Diego/Results

[Home](https://2019.igem.org/Team%3ACCA_San_Diego)

Team

[Team Members](https://2019.igem.org/Team%3ACCA_San_Diego/Team)
[Collaborations](https://2019.igem.org/Team%3ACCA_San_Diego/Collaborations)

Project

[Description](https://2019.igem.org/Team%3ACCA_San_Diego/Description)
[Design](https://2019.igem.org/Team%3ACCA_San_Diego/Design)
[Experiments](https://2019.igem.org/Team%3ACCA_San_Diego/Experiments)
[Notebook](https://2019.igem.org/Team%3ACCA_San_Diego/Notebook)
[Contribution](https://2019.igem.org/Team%3ACCA_San_Diego/Contribution)
[Results](https://2019.igem.org/Team%3ACCA_San_Diego/Results)
[Attributions](https://2019.igem.org/Team%3ACCA_San_Diego/Attributions)

Parts

[Parts Overview](https://2019.igem.org/Team%3ACCA_San_Diego/Parts)
[Basic Parts](https://2019.igem.org/Team%3ACCA_San_Diego/Basic_Part)
[Part Collection](https://2019.igem.org/Team%3ACCA_San_Diego/Part_Collection)

[Safety](https://2019.igem.org/Team%3ACCA_San_Diego/Safety)

Human Practices

[Human Practices](https://2019.igem.org/Team%3ACCA_San_Diego/Human_Practices)
[Education and Engagement](https://2019.igem.org/Team%3ACCA_San_Diego/Public_Engagement)

Awards

[Medal Criteria](https://2019.igem.org/Team%3ACCA_San_Diego/Medal_Criteria)
[Entrepreneurship](https://2019.igem.org/Team%3ACCA_San_Diego/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3ACCA_San_Diego/Hardware)
[Measurement](https://2019.igem.org/Team%3ACCA_San_Diego/Measurement)
[Model](https://2019.igem.org/Team%3ACCA_San_Diego/Model)
[Software](https://2019.igem.org/Team%3ACCA_San_Diego/Software)

[Judging](https://igem.org/2019_Judging_Form?team=CCA_San_Diego)

# Results

Clones containing the chrysene degradation construct were able to successfully use chrysene for metabolism. Experimental settings were designed so that chrysene was the only available carbon source. As you can see in the graph, whereas the control strain was unable to utilize chrysene as a carbon source, the strain in which Part:BBa K2566006 was cloned is able to metabolize chrysene and grow using chrysene as a sole carbon source. BL21 cells were used in this graph.

![](https://static.igem.org/mediawiki/2019/7/71/T--CCA_San_Diego--ChryseneBiotransformation.png)
HSU-ETCD was assembled into PSB1C3 using Gibson assembly. You can see the Experiments tab for more information on Gibson primers and colony PCR primers that correspond to this part. To measure the amount of hydrogen produced, H2Blue, a reagent that is blue and becomes clear in the presence of dissolved hydrogen, was used; lower OD650 on this graph corresponds to lower levels of blue. Compared to the control strain, the strain in which HSU-ETCD was cloned exhibits higher amounts of hydrogen production. The strain used in this graph is NEB 5alpha.

![](https://static.igem.org/mediawiki/2019/6/6b/T--CCA_San_Diego--HSUETCD.jpeg)