iGEM 2020 — Team Baltimore_BioCrew

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# Team:Baltimore BioCrew/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Baltimore BioCrew/Engineering

2020 Baltimore Biocrew

![Safety Header](https://static.igem.org/mediawiki/2020/6/69/T--Baltimore_BioCrew--EngineeringHeader2.png)

# Engineering

We introduced iron uptake and processing genes from other bacteria into Synechococcus sp. CB0101, to make Synechococcus more adaptable to a low-iron environment. Click each section to learn more!

* Research
  *arrow\_downward*

  We read numerous pieces of literature and interviewed experts and stakeholders in the field.

  Our New Part team’s primary piece of literature was by  [Alghren et al.](https://sfamjournals.onlinelibrary.wiley.com/doi/full/10.1111/1462-2920.14893) It contains a list of genes found in cyanobacteria that evolved to live in low-iron environments. It shows the different genes, which climates they are most prominent, and which organisms evolved them. It also briefly describes the effect of the genes and why they would be evolutionarily advantageous. We used this to find genes that appeared to have a correlation to iron efficiency.

  We also conducted multiple interviews with experts in related fields. More information is available on our  [Human Practices page](https://2020.igem.org/Team%3ABaltimore_BioCrew/Human_Practices). Gabriel Browning is affiliated with the University of South Florida, and is a researcher on iron ligands. His input was invaluable in building our understanding of iron dynamics in the ocean.

  Dr. Jim George spoke to the team about the ecology of the Chesapeake Bay, where our strain of cyanobacteria (Synechococcus CB1010) was found and on which our hometown of Baltimore lies. Dr. George was a senior policy advisor at Water and Science Administration, Maryland Department of Environment—he worked for 12 years for the Water Quality Restoration and accountability program, which implements policy to regulate loads put in water and established Maryland’s Watershed Implementation Plan for the Chesapeake Bay. Currently, he does development for the Water and Science Administration and development of a statewide water resources plan. His administrative experience and knowledge of stakeholders and policy related to our project.

  We consulted Dr. Allen Pace for information about iron uptake in phytoplankton. Dr. Place was a professor at the local University of Maryland Institute of Marine & Environmental Technology. He focuses on molecular mechanisms and their interactions with organisms and the environment. His knowledge aided in the development of our project, by helping further explain the behavior of phytoplankton with iron and factors that affect phytoplankton concentration in its environment.

  Donald A. Brown is a bioethicist of some renown. He is currently the scholar in residence for Sustainability Ethics and Law through Widener’s Environmental Law and Sustainability Center. Previously, he taught at Penn State University and was a project manager for the EPA’s Office of International Environmental Policy. He also represented the EPA in UN delegations concerning climate change and ethics. He helped us ask the appropriate ethical questions about our project.

  James J. Johnson is the Executive Director of the Deep Sea Fisherman’s Union. He spoke to us about the human implications of our project; the communities and economies that it would affect and the ways that it could serve the people.

* Imagine
  *arrow\_downward*

  [The decline of phytoplankton has been steady for decades,](https://www.scientificamerican.com/article/phytoplankton-population/) and poses an enormous risk to all of Earth’s ecosystems, with ripple effects throughout both the food chain and the carbon cycle. Since phytoplankton are small organisms and many are easily modified, we thought there might be a synthetic biology experiment that could address this global problem.
  By increasing phytoplankton’s effectiveness with using iron, we hope to increase the rates of photosynthesis to sequester more carbon dioxide and help slow down the effects of climate change.

* Design
  *arrow\_downward*

  We selected the following genes because the Ahlgren et al paper found that they were present in extra copies in cyanobacteria which have evolved to live in low-iron environments (click on gene name for a link to the registry page):
  + [isiB (Flavodoxin):](http://parts.igem.org/Part%3ABBa_K3651000) Low-potential electron donor for redox enzymes
  + [ZupT:](http://parts.igem.org/Part%3ABBa_K3651001) Fe2+ transport gene
  + [idiA:](http://parts.igem.org/Part%3ABBa_K3651002) Fe3+ import gene
  + [feoA:](http://parts.igem.org/Part%3ABBa_K3651003) Transition metal ion binding gene
  + [feoB:](http://parts.igem.org/Part%3ABBa_K3651004) GTP-driven Fe2+ uptake
  + [tonB](http://parts.igem.org/Part%3ABBa_K3651005) Siderophore transport gene
  + [bfr (Ferritin):](http://parts.igem.org/Part%3ABBa_K3651027) Iron storage protein
  We are trying to combine each of these proteins into a single strain of cyanobacteria to make it extraordinarily efficient at using iron. It will then be even better at surviving in the low iron areas of the ocean where phytoplankton are rare.

![](https://static.igem.org/mediawiki/2020/e/e5/T--Baltimore_BioCrew--MicroTubes.jpeg)

![](https://static.igem.org/mediawiki/2020/8/8e/T--Baltimore_BioCrew--AlgaeTubes.jpeg)

* Build
  *arrow\_downward*

  The steps that we used to design our parts was:
  + Identified different cyanobacteria genes involved in iron-related cellular processes from "Genomic Mosaicism Underlies the Adaptation of Marine Synechococcus Ecotypes to Distinct Oceanic Iron Niches" by Ahlgren et al.
  + Determined if those genes were already present in our strain CB0101.
  - Found the genes from the paper in the PCC6803 reference strain of cyanobacteria and found their amino acid sequences on UnitProt.
  - Put each amino acid sequence into a standard NCBI protein BLAST search and chose a search set for “Synechococcus sp. CB0101 (taxid:232348).”
  * This switch was performed because the PCC6803 strain was too expensive, and the CB0101 strain was already available in our lab.- Found the query cover percentage of the matched gene in CB0101 compared to the PCC6803 strain.
  - Any gene that had a query cover percentage that was high wasn’t chosen as CB0101 already had it.
  - The DNA sequences of the remaining genes were found by putting the amino acid sequence into the codon optimization tool from IDT and having it calculate the optimized sequence for Synechococcus.+ Added additional regulatory sequences from the 2016 Edinburgh team (who also expressed genes in cyanobacteria) were added:
  - Promoter: J23105
  - RBS: B0034
  - Terminator: B0015+ Ordered genes as gBlocks from IDT
  + Digested gBlocks and pSB1C3 vector(with RFP gene insert) with EcoRI and PstI (protocols can be found at our  [Protocols](https://2020.igem.org/Team%3ABaltimore_BioCrew/Experiments) page.+ Ligated vector and insert together and transformed into *E. coli*
    + Found *E.coli* that had successfully transformed, as indicated by absence of RFP
    + Picked out colonies and performed colony PCR

* Learn
  *arrow\_downward*

  ![](https://static.igem.org/mediawiki/2020/d/d9/T--Baltimore_BioCrew--NewPartLab2.jpeg)
  We got into the lab late and thus only had time for one set of genes. After digest, ligation, transformation, and culture, our goal was to isolate a positive clone from any of the successful cultures of our genes in *E. coli,* which we chose to use to clone our plasmids for future use in cyanobacteria. After several iterations of colony screening, we detected positive clones for all of our genes.

  Our next steps would be:
  + Clone the genes in *E. coli.*
  + Transfer the genes to cyanobacteria by transformation; at this point each sample would only receive one new gene.
  + Test cyanobacteria with each individual gene in low iron concentrations (starting, perhaps, at 1/100 normal concentration). Measure by OD after 2 weeks and see which grow better than starting strain. Do not move forward with those that show no change.
  + For those that show improved growth, introduce the genes in varying combinations into cyanobacteria We will need to put them into different plasmids so that they are resistant to different antibiotics. Test to see which in combination strains grow better than starting strain.
  + Repeat, consolidating as many genes as show an effect into a single strain.

* Improve
  *arrow\_downward*

  We found positive clones for all of our genes--although some more strongly than others. We can confirm that transformation was successfully completed, and now we can go on to test for functionality.

  In the future, we want to test our genes in cyanobacteria. We would like to determine the growth rates of each modified strain in media of various iron concentrations and compare it to the many control samples we have already taken; do the new genes work better? Are there some that might do better as a combination? Can we create a truly optimized strain of cyanobacteria?

  Some of our genes are part of the same pathways--for example, feoA and feoB are linked--and would likely perform better if they were added together. When we test all of the genes individually, if one works poorly on its own it might get written off too early; however, it would be impractical to test every possible combination of genes. A potential solution is to find a pair that works well together, and then try adding back the less successful genes to see if they make an impact.

  After that, the research phase begins again as we search for specific uses for our product. While we cannot use it in the oceans directly, an efficient strain of cyanobacteria could be useful to both research and industry.

### Results

After several trials of colony screening, we obtained at least one successful colony from each of our genes! These gels displays the results obtained. (It should be noted that the gel for Sample 1 was old, and some of the samples may have been lost into the buffer solution.)

![](https://static.igem.org/mediawiki/2020/9/9d/T--Baltimore_BioCrew--NewPartGel.jpeg)

A project by the Baltimore BioCrew 2020 team.

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# Team:Baltimore BioCrew/Experiments

2020 Baltimore Biocrew

![Protocols Header](https://static.igem.org/mediawiki/2020/d/de/T--Baltimore_BioCrew--ProtocolsHeader2.png)

# Protocols

### Protocol for Growing *Synechococcus CB0101* with Variable Iron Concentrations

[Growth Protocol](https://static.igem.org/mediawiki/2020/1/1f/T--Baltimore_BioCrew--GrowthProtocol.pdf)

### Protocol for Measuring Red Fluorescent Protein to Test Efficacy of pFUR Promoter Contstructs in *E.coli*

[Promoter Measurement Protocol](https://static.igem.org/mediawiki/2020/7/72/T--Baltimore_BioCrew--MeasureRFPProtocol.pdf)

### Digest Protocol

[Digest Protocol](https://static.igem.org/mediawiki/2020/3/3c/T--Baltimore_BioCrew--DigestProtocol.pdf)

### Ligation Protocol

[Ligation Protocol](https://static.igem.org/mediawiki/2020/2/21/T--Baltimore_BioCrew--LigationProtocol.pdf)

A project by the Baltimore BioCrew 2020 team.

##### Contact

*mail\_outline*
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##### Follow Us!

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# Team:Baltimore BioCrew/Results

2020 Baltimore Biocrew

![Results Header](https://static.igem.org/mediawiki/2020/d/d0/T--Baltimore_BioCrew--ResultsHeader4.png)

# Results Overview

#### Bronze Medal Criteria

| Link to page | Summary of how we fulfilled the criteria |
| --- | --- |
| [Project Description](https://2020.igem.org/Team%3ABaltimore_BioCrew/Description) | The Baltimore BioCrew was inspired to design a project to increase growth of phytoplankton to stabilize the food supply for the marine food chain and absorb CO2 from the atmosphere to reduce climate change. Out of the many organisms that are phytoplankton, we chose a strain of cyanobacteria (Synechococcus CB0101) and decided to make it better at growing in environments with low iron. |
| [Attributions](https://2020.igem.org/Team%3ABaltimore_BioCrew/Attributions) | To tackle all the parts of our project, our team of high school students worked in many small groups. We had three groups who planned experiments that involved being in our lab and five groups who were responsible for tasks that could be done outside of the lab. |
| [Contribution](https://2020.igem.org/Team%3ABaltimore_BioCrew/Contribution) | Our first wet lab groups characterized the growth of our strain of cyanobacteria under varying iron concentrations and contributed validation of an inexpensive measurement technique that we had not seen used by any other iGEM teams. |

#### Silver Medal Criteria

| Link to page | Summary of how we fulfilled the criteria |
| --- | --- |
| [Engineering Success](https://2020.igem.org/Team%3ABaltimore_BioCrew/Engineering) | Our second wet lab group identified seven genes involved in iron-related cellular processes from strains of cyanobacteria that evolved in low iron environments. The team optimized these genes for expression in Synechococcus CB0101 to engineer a single strain of cyanobacteria to be super efficient at using iron ([BBa\_K3651030](http://parts.igem.org/Part%3ABBa_K3651030), [BBa\_K3651011](http://parts.igem.org/Part%3ABBa_K3651011), [BBa\_K3651012](http://parts.igem.org/Part%3ABBa_K3651012), [BBa\_K3651032](http://parts.igem.org/Part%3ABBa_K3651032), [BBa\_K3651014](http://parts.igem.org/Part%3ABBa_K3651014), [BBa\_K3651015](http://parts.igem.org/Part%3ABBa_K3651015), [BBa\_K3651033](http://parts.igem.org/Part%3ABBa_K3651033)). |
| [Collaborations](https://2020.igem.org/Team%3ABaltimore_BioCrew/Collaborations) | The collaboration team coordinated with multiple iGEM teams to create peer-reviewed proceedings for iGEM 2020, design a class to raise awareness about human impact on aquatic ecosystems, and record a podcast to communicate our project to the public. |
| [Human Practices](https://2020.igem.org/Team%3ABaltimore_BioCrew/Human_Practices) | The human practices team arranged interviews with many experts including a research student who studies iron cycling in the ocean, a local professor whose expertise is adaptations of marine organisms, an environmental policy advisor who protects watersheds at the state level, a scholar in environmental ethics who has negotiated climate change issues with the United Nations, and a fisherman who livelihood is impacted by climate and marine ecosystems. |
| [Proposed Implementation](https://2020.igem.org/Team%3ABaltimore_BioCrew/Implementation) | The human practices team considered how our project could be implemented in the real world. They identified communities who would be interested in increasing phytoplankton populations such as scientists who want to address climate change by increasing photosynthesizing organisms which consume carbon dioxide. They considered the ethical challenges and restrictions when releasing engineered organisms. They identified ways to avoid negative environmental impacts such as growing engineered phytoplankton on farms instead of releasing into marine environment and also working with the state Department of Environment to have the appropriate permits and certification. |

#### Gold Medal Criteria

| Link to page | Summary of how we fulfilled the criteria |
| --- | --- |
| [Improvement of an Existing Part](https://2020.igem.org/Team%3ABaltimore_BioCrew/Improve) | Our third wet lab group addressed the question of how to keep our engineered cyanobacteria from growing too much and causing algae blooms. They set out to improve the sensitivity of iron-sensing promoters. If these promoters were used upstream of genes that promote cell death, then the cyanobacteria population would stop increasing once the iron concentration needed to activate the promoter is reached. The groups designed a set of iron-sensing promoters with additional repeats of the FUR box ([BBa\_K3651020](http://parts.igem.org/Part%3ABBa_K3651020), [BBa\_K3651021](http://parts.igem.org/Part%3ABBa_K3651021), [BBa\_K3651023](http://parts.igem.org/Part%3ABBa_K3651023), [BBa\_K3651024](http://parts.igem.org/Part%3ABBa_K3651024)) which improved upon promoters from the 2018 ECUST iGEM team ([BBa\_K2737003](http://parts.igem.org/Part%3ABBa_K2737003), [BBa\_K2737004](http://parts.igem.org/Part%3ABBa_K2737004), and [BBa\_K2737005](http://parts.igem.org/Part%3ABBa_K2737005)). |
| [Integrated Human Practices](https://2020.igem.org/Team%3ABaltimore_BioCrew/Human_Practices) | The interviews with experts and stakeholders help shape our project design and science communication initiatives. Discussions with scientists helped us identify which phytoplankton we should attempt to modify and gave us an understanding of how phytoplankton use iron so we could identify which genes to utilize. Also talking with scientists, policymakers, and stakeholders about the science and impact of climate change encouraged us to educate people about climate and marine environments through social media and a class. |
| [Math Modeling](https://2020.igem.org/Team%3ABaltimore_BioCrew/Model) | The math modeling team used the Monod Model for substrate-limited cell growth to model the growth of cyanobacteria as iron concentration changes. They optimized the parameters in the model using data from the literature. This team analyzed their optimization results to improve upon the design of their experiment to measure cyanobacteria growth at their homes. They plan to use this model to compare the kinetic parameters of our engineered cyanobacteria and identify which genes increase growth best. |
| [Proof Of Concept](https://2020.igem.org/Team%3ABaltimore_BioCrew/Proof_Of_Concept) | In order to justify the basis for our project, one of our wet lab teams tracked the growth of cyanobacteria in various concentrations of iron. They observed that as the concentration of Fe(II) increases in the media there is an increased growth of *Synechococcus CB0101*. The results provide proof of concept for the whole project because they demonstrate that our decision to engineer a *Synechococcus* strain with increased access to iron will likely increase the growth of the cyanobacteria. |
| [Science Communication](https://2020.igem.org/Team%3ABaltimore_BioCrew/Education) | Through social media, the human practices team communicated to the public about synthetic biology, phytoplankton, and climate change by posting a series of stories that included polls and questions that increased engagement with our accounts. This team also developed a class on oceans, phytoplankton, and climate change, which we taught to middle school students. |

A project by the Baltimore BioCrew 2020 team.

##### Contact

*mail\_outline*
info@bugssonline.org

##### Follow Us!

* [@bugsslab](https://twitter.com/bugsslab?lang=en)
* [bugsslab](https://www.instagram.com/bugsslab/)
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