Team:Baltimore BioCrew/Design

2020 Baltimore Biocrew


Project Design

Design

Our project modifies cyanobacteria, Synechococcus CB0101, as described in Ahlgren et. al. We believe that bringing together the mutations that have evolved separately into one strain will enhance the ability of phytoplankton to grow in different ocean conditions that have a low abundance of iron. The increased iron levels will increase photosynthesis in phytoplankton, which will stabilize the marine food chain and absorb CO2 from the atmosphere.


Characterization of Cyanobacteria Growth in Iron

To test the significance of iron amounts to the phytoplankton population, we used Synechococcus CB0101 (initial OD 0.05) and put it into 5 conical tubes with SN media with varying amounts of iron concentrations: No iron, 0.01X, 0.1X, 1X (0.023 mM), and 10X normal iron concentration. The growth of these tubes were measured by optical density.


Improving Iron Consumption Efficiency in Cyanobacteria

To improve the ability of Synechococcus (a genus of cyanobacteria) to live in a low-iron environment, we decided to try adding iron uptake and processing genes from naturally evolved species of cyanobacteria to our isolate of Synechococcus CB0101 . Highly expressed iron consumption genes were selected from Ahlgren et al. 2019 [1] that are associated with iron uptake in iron-deficient environments. Our reasoning was that if we add more pathways for iron uptake, the bacteria will be able to uptake a greater amount of the iron from the environment as iron consumption pathways may be enhanced to process different oxidation states of iron or different iron-containing molecules.

The paper described a number of genes, and 7 of the genes were chosen and cloned in our bacteria (see table below) Ahlgren et al. describes several genes related to iron consumption that have been evolved in iron-deficient environments. Separate samples of E. coli cells were modified with each of these genes based on the findings of the Ahlgren study. These genes are: feoA, feoB, isiB, idiA, pcopM, tonB, and zupT.


  • Gene Names and Functions arrow_downward
    Gene Names Function
    feoA Transition metal binding ion, works in complex with feoB
    feoB Transmembrane transporter of a GTP-driven Fe2+ uptake system
    isiB Encodes for Flavodoxin, which functions as an electron donor in redox reactions
    idiA Metal binding ion, protects against oxidative damage
    pcopM Encodes for Ferritin, an iron storage protein
    tonB Allows for siderophore uptake across the membrane
    zupT Mediates uptake of divalent cations and Fe2+

We added a promoter, ribosome binding site (RBS), and terminator to each before ordering them to be synthesized. The basic genetic parts we used to build the composite parts around our genes were based on work of the 2016 Edinburgh iGEM team. The figure below show our general construct design, where the purple box indicates the location where we inserted one of the following genes: feoA, feoB, isiB, idiA, pcopM, tonB, or zupT.


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  • References
    1. Ahlgren, N.A., Belisle, B.S. and Lee, M.D. (2020), Genomic mosaicism underlies the adaptation of marine Synechococcus ecotypes to distinct oceanic iron niches. Environ Microbiol, 22: 1801-1815. doi:10.1111/1462-2920.14893