Team:Lethbridge/Parts

Overview

The criteria used to decide on which peptides to use for our project was based on specific properties, including the following:

  • Small Size
  • Net charge
  • Lack of Toxicity to Humans
  • Specificity or Select Range of Function
  • Heat Stability

By choosing these properties, we can select appropriate parts required for building a functional construct, as well as optimize the production and functionality of the peptides.

Already Existing Parts

Basic Parts

Our E.coli constructs were designed using Biobrick assembly. However, Golden Gate restriction sites were also added on both sides of the AMP coding sequence in each part. If the construct is cut in those locations, the AMP sequence will be removed, and can then be inserted into one of our plant constructs. This allows us to exchange the AMPs in our four plant constructs with any of the AMPs in our many E.coli constructs if needed.

The one exception to the design described above is our BMAP-18 E.coli Insert, which was the first sequence that we ordered. As it was ordered before all elements of our construct design were finalized, it does not contain the BsaI restriction sites on either side of the AMP sequence. However, since one of our plant constructs contains BMAP-18, we will still be able to express BMAP-18 in both potato plants and E.coli.

Composite Parts for E. coli

Our potato constructs were designed using Golden Gate assembly, with Biobrick restriction sites on either end of the construct. The Golden Gate restriction sites between each part allow for the constructs to be cut apart and reassembled, facilitating the exchange of parts, particularly the AMP coding sequence. The Biobrick restriction sites can be used to cut at the ends of the construct so that each of the individual parts remain together. The constructs also contain a left-border T-DNA repeat before the promoter and a right-border T-DNA repeat after the terminator, so that they are able to be taken up by Agrobacterium tumefaciens for use in agrobacterium-mediated transformation of the potato plants.

Composite Parts for Potato Plants

Citations

  1. Wang, G. (2020). The Antimicrobial Peptide Database. Retrieved 2020.
  2. Mardirossian, M., Pompilio, A., Degasperi, M., Runti, G., Pacor, S., Bonaventura, G. D., & Scocchi, M. (2017). D-BMAP18 Antimicrobial Peptide Is Active In vitro, Resists to Pulmonary Proteases but Loses Its Activity in a Murine Model of Pseudomonas aeruginosa Lung Infection. Frontiers in Chemistry, 5. doi:10.3389/fchem.2017.00040
  3. Fujimura, M., Ideguchi, M., Minami, Y., Watanabe, K., & Tadera, K. (2004). Purification, Characterization, and Sequencing of Novel Antimicrobial Peptides,Tu-AMP 1 andTu-AMP 2, from Bulbs of Tulip (Tulipa gesnerianaL.). Bioscience, Biotechnology, and Biochemistry, 68(3), 571-577. doi:10.1271/bbb.68.571