Team:Baltimore BioCrew/Engineering

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. 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. 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, 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): Low-potential electron donor for redox enzymes
  • ZupT: Fe2+ transport gene
  • idiA: Fe3+ import gene
  • feoA: Transition metal ion binding gene
  • feoB: GTP-driven Fe2+ uptake
  • tonB Siderophore transport gene
  • bfr (Ferritin): 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.

• 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 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

  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.)