Team:OhioState/Design

R. palustris, A. vinelandii, and P. stutzeri were all initially considered to be donors for their nitrogenase genes^[1][2][3][5]. Ultimately, R. palustris was chosen because of its relative ease of access, genetic similarity to recipients, and contiguous nitrogenase island. We started with PCR to amplify the genes using multiple primer pairs. Because of limitations on polymerase ability to replicate such long amplicons, we broke the nif cluster into four sections:

The first fragment (10286bp) included nifQUSVW (Molybdenum cofactor transport and biosynthesis proteins), fixABCX(electron transport proteins), and other miscellaneous FeS proteins.

The second fragment (3341bp + 4079bp) contained nifENX, which provide metal cofactor assembly scaffolding, and nifHDK, the structural subunits of nitrogenase specialized for the molybdenum cofactor. Both nifENX and the whole fragment were amplified.

The third fragment (9736bp) included the nifA transcriptional activator protein, nifBZT, ferredoxin/FeS proteins, and other uncharacterized proteins.

Three additional fragments were made to also amplify out the vanadium and iron-only nitrogenase gene clusters also native to R.palustris. They contain homologous genes specialized for different cofactors.

The appropriate fragments were ligated into the vector backbone mini-CTX1, which contains integrase sequences from Pseudomonas. Once transferred into recipient pseudomonas strains, the insert integrates into the genome.^[4]

Integration of mini-CTX1 into Pseudomonas Chromosome

Concurrently, we used pULB113, an RP4::mini-Mu plasmid, for the isolation of these clusters. It encodes for a replicative transposon that self-excises parts of a donor genome into plasmids, those containing functional nif clusters which can then be mated into recipient strains and selected for on nitrogen limited media^[5].

Genome excision and transfer by pULB113

At this point, this is where we stopped all lab work to complete our Wiki by the deadline. After the Jamboree, we plan to follow up with the below plan.

Once on plasmids, these new clusters will be mated triparentally into recipients Pseudomonas putida KT2440, CT364, and Pseudomonas protegens PF5, chosen for their natural ability to colonize cereal crops^[6][7]. Seedlings can be inoculated with bacterial culture and allowed to germinate on both agar and in soil. Intercellular presence of the engineered strains in the roots of corn would allow for both a microaerophilic environment ideal for the aerobic growth of the bacterium and low oxygen concentrations for the function of nitrogenase. Colonization could be confirmed with DAPI staining of seedling root tissue[8], and indirectly by measuring plant growth promotion. Seed inoculation is not a new practice: general plant growth promoting bacteria have been commercialized in a similar manner in previous years as an in-furrow biostimulant.^[9][10]

References

[1] Desnoues, N. (2003). Nitrogen fixation genetics and regulation in a Pseudomonas stutzeri strain associated with rice. Microbiology, 149(8), 2251–2262. https://doi.org/10.1099/mic.0.26270-0

[2] Yan, Y., Yang, J., Dou, Y., Chen, M., Ping, S., Peng, J., … Jin, Q. (2008). Nitrogen fixation island and rhizosphere competence traits in the genome of root-associated Pseudomonas stutzeri A1501. Proceedings of the National Academy of Sciences, 105(21), 7564–7569. https://doi.org/10.1073/pnas.0801093105

[3] Oda, Y., Samanta, S. K., Rey, F. E., Wu, L., Liu, X., Yan, T., … Harwood, C. S. (2005). Functional Genomic Analysis of Three Nitrogenase Isozymes in the Photosynthetic Bacterium Rhodopseudomonas palustris. Journal of Bacteriology, 187(22), 7784–7794. https://doi.org/10.1128/JB.187.22.7784-7794.2005

[4] Tung T Hoang, Alecks J Kutchma, Anna Becher, Herbert P Schweizer, Integration-Proficient Plasmids for Pseudomonas aeruginosa: Site-Specific Integration and Use for Engineering of Reporter and Expression Strains, Plasmid, Volume 43, Issue 1, 2000, Pages 59-72, ISSN 0147-619X, https://doi.org/10.1006/plas.1999.1441.

[5] Van Gijsegem, F. (2018). Use of RP4::Mini-Mu for Gene Transfer. Methods in Molecular Biology (Clifton, N.J.), 1681, 287–302. https://doi.org/10.1007/978-1-4939-7343-9_21

[6] Setten, L., Soto, G., Mozzicafreddo, M., Fox, A. R., Lisi, C., Cuccioloni, M., … Ayub, N. D. (2013). Engineering Pseudomonas protegens Pf-5 for nitrogen fixation and its application to improve plant growth under nitrogen-deficient conditions. PloS One, 8(5), e63666. https://doi.org/10.1371/journal.pone.0063666

[7] Planchamp, C., Glauser, G., & Mauch-Mani, B. (2015). Root inoculation with Pseudomonas putida KT2440 induces transcriptional and metabolic changes and systemic resistance in maize plants. Frontiers in Plant Science, 5. https://doi.org/10.3389/fpls.2014.00719

[8] Newcastle iGEM 2018. (n.d.). Team:Newcastle—2018.igem.org. Retrieved June 12, 2019, from Alternative Roots website: https://2018.igem.org/Team:Newcastle

mkulwiec. (n.d.). 3Bar Biologics. Retrieved October 19, 2019, from 3 Bar Biologics website: http://www.3barbiologics.com/

[10] Pivot Bio. (n.d.). Retrieved October 19, 2019, from https://www.pivotbio.com