Team:Rochester/Parts

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Parts Overview

Parts Table

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Parts

Parts Overview

All our parts were synthesized as gBlocks by IDT with the exception of oprf which was synthesized by Twist Bioscience. Most of the sequences for the genes were obtained from our chassis organism Shewanella oneidensis MR-1 while the others were obtained from Pseudomonas aeruginosa or Escherichia coli MP021561.2 and codon optimized for S. oneidensis MR-1. Our parts were assembled into our backbone pcD8 (Keitz Lab, University of Texas Austin)¹ for expression in S. oneidensis MR-1 using the Golden Gate cloning method, where our parts were already synthesized to include the Golden Gate primers. We created an aptamer collection, which consists of five single stranded DNA aptamers, with corresponding part numbers: BBa_K3724001 - BBa_K3724006 (BBa_K3724002 has been deleted). We went through several design-build-test cycles for the asymmetric PCR conditions, in order to optimize the percentage and concentration of ssDNA. We obtained an exceptionally high ssDNA:dsDNA ratio of 7:1 for all optimized aptamers, and concentrations in the 7 - 90 µg/mL range. This part collection can be used to selectively inhibit signaling of three pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), as well as acute immune response protein CRP, and antimicrobial peptide lactoferrin. With detection limits in picomolar range, our aptamer collection can serve as a useful tool for other iGEM teams because each aptamer can be part of potential treatments for various diseases that result in an overactive immune response. Currently it is used in the detection of biomarkers for sepsis diagnosis. Moreover, the molecules our aptamers target are important in cancer, so the aptamer collection can also be part of a cancer detection tool. They comply with all RFC standards of interest to iGEM, so they can also be combined with other biobricks and be delivered together with other constructs.

Parts Table

Favorite	Name	Type	Description	Designer	Length
	BBa_K3724001	Basic	IL-6 aptamer	Anca Gabriela Frasineanu	57
	BBa_K3724003	Basic	TNF-α aptamer	Anca Gabriela Frasineanu	25
	BBa_K3724004	Basic	CRP aptamer	Anca Gabriela Frasineanu	44
❤	BBa_K3724005	Basic	Lactoferrin Aptamer	Anca Gabriela Frasineanu	59
	BBa_K3724006	Basic	IL-1β aptamer	Anca Gabriela Frasineanu	30
	BBa_K3724008	Coding	Diguanylate cyclase YdeH	Tracey Moyston	921
	BBa_K3724009	Coding	Tetraheme cytochrome c protein/ quinol dehydrogenase cymA	Tracey Moyston	593
	BBa_K3724010	Coding	Riboflavin biosynthesis protein RibF	Tracey Moyston	966
	BBa_K3724011	Coding	Outer Membrane Porin OprF	Tracey Moyston	1083
❤	BBa_K3724015	Composite	Riboflavin synthesis gene cluster	Tracey Moyston	3564

References

1. ACS Synth. Biol. 2020, 9, 9, 2301–2315Publication Date:July 28, 2020https://doi.org/10.1021/acssynbio.9b00517