Team:Lund/Parts
Parts are crucial for the spreading of knowledge in iGEM. Parts are DNA sequences that teams share, which can help with research outside of iGEM. Team Lund 2021 is submitting parts coding for proteins and peptides designed to inhibit the bacterial amyloid curli. Additionally, we are submitting parts coding for these inhibitors connected to signal peptides for secretion by Limosilactobacillus reuteri. All the parts are codon-optimized for L. reuteri. During 2021, Team Lund worked with the parts seen bellow
CsgC: BBa_K3955000
The protein Curli assembly protein CsgC (henceforth called CsgC) is a natural part of the curli production system; it is a chaperone that prevents fibril formation in the periplasm. It has been shown in vitro to efficiently inhibit curli formation from CsgA monomers [1].
PeptideNN17R: BBa_K3955050
The protein CsgF is also a natural part of the curli production system, which binds to the CsgG pore on the outer plasma membrane. CsgG transports the CsgA monomers outside of the cell, while CsgF may assist CsgA in binding to the nearest curli fibril. This peptide is derived from CsgF, but one residue has been changed to a large arginine; This causes the peptide to block the pore, hindering CsgA from exiting [2].
As it is only 17 amino acid residues long, we expected to have difficulties with secretion. Therefore the part is designed as 5 repeats of the peptide, with alanine added in between each repeat. The alanine makes a cleavage site for pancreatic elastase, which is found in the gut.
The sequence was reverse translated from the amino acid sequence in the original article, then codon optimized using a codon optimizer tool. This was followed by manual adjustments to minimize repeats and high complexity for synthesising.
DegP: BBa_K3955100
Periplasmic serine endoprotease DegP (henceforth written DegP) is a protease with amyloid specificity [3]. Bacteria which secrete DegP have been shown to out compete E. coli biofilm formation, while knock out mutants of the same strain may not [4] As curli is a major component of E. coli biofilms and is amyloidic, DegP is a viable curli inhibitor candidate [5].
DB3DB3 and slpMOD: BBa_K3955101
DB3DB3 is a peptide made to inhibit amyloid beta, but we were hoping to test its effect on CsgA [6]. Similarly to PeptideNN17R, we were expecting issues with secretion and therefore designed the part as 3 repeats of the peptide. Alanine was added in between repeats here as well to create a cleavage site for pancreatic elastase which may be found in the gut.
The sequence was reverse translated from the amino acid sequence in the original article, then codon optimized using a codon optimizer tool. This was followed by manual adjustments to minimize repeats and high complexity for synthesising.
slpMOD is an existing part in the registry, BBa_K3183008, and has been studied previously in L. lactis.
DB3DB3 and Amyl: BBa_K3955102
Amyl is a signal peptide originating from a heat- and pH-stable α-amylase of Bacillus licheniformis [7]. It has been shown to successfully cause secretion in L. reuteri when fused with GFP [8].
DB3DB3 and Usp 45: BBa_K3955103
Usp45 originates in Lactococcus lactis and is a major extracellular component [9]. It’s secretion signal peptide has been shown to successfully cause secretion in L. reuteri when fused with GFP [8].
DB3DB3 and Mub: BBa_K3955104
Mub originates from a mucus adhering cell surface protein found in L. reuteri [10]. The sequence has previously been shown to induce secretion when fused to GFP and recombinantly expressed in L. reuteri [8].
tANK6 and slpMOD: BBa_K3955105
tANK6 is a peptide made to inhibit amyloid beta, we wanted to investigate what impact tANK6 could have on CsgA [11]. Issues with secretion were expected for tANK6 as well and was therefore designed with 3 repeats of the peptide. Alanine was added in between repeats here as well to create a cleavage site for pancreatic elastase which may be found in the gut.
The sequence was constructed manually from the amino acid sequence in the original article, amino acid codon tables and a codon frequency table for L. reuteri.
1. Evans M, Chorell E, Taylor J, Åden J, Götheson A, Li F et al. The Bacterial Curli System Possesses a Potent and Selective Inhibitor of Amyloid Formation. Molecular Cell. 2015;57(3):445-455.
2. Zhaofeng Yan, Meng Yin, Jianan Chen, Xueming Li. Assembly and substrate recognition of curli biogenesis system. Nature Communications [Internet]. 2020 Jan 1 [cited 2021 Oct 9];11(1):1–10. Available from: https://search.ebscohost.com/login.aspx?direct=true&db=edsdoj&AN=edsdoj.19f841225ae740398e4682f0391a5534&site=eds-live&scope=site
3. Krojer T, Sawa J, Schäfer E, Saibil H, Ehrmann M, Clausen T. Structural basis for the regulated protease and chaperone function of DegP. Nature. 2008;453(7197):885-890.
4. Fang K, Jin X, Hong SH. Probiotic Escherichia coli inhibits biofilm formation of pathogenic E. coli via extracellular activity of DegP. Scientific Reports [Internet]. [cited 2021 Oct 9];8(1). Available from: https://search.ebscohost.com/login.aspx?direct=true&db=edselc&AN=edselc.2-52.0-85044277078&site=eds-live&scope=site
5. Barnhart, M. M., & Chapman, M. R. (2006). Curli Biogenesis and Function. Annual Review of Microbiology, 60(1), 131–147. doi:10.1146/annurev.micro.60.080805.142106
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838481/
6. Perov S, Lidor O, Salinas N, Golan N, Tayeb- Fligelman E, Deshmukh M et al. Structural Insights into Curli CsgA Cross-β Fibril Architecture Inspire Repurposing of Anti-amyloid Compounds as Anti-biofilm Agents. PLOS Pathogens. 2019;15(8):e1007978.
7. YUUKT T, NOMURA T, TEZUKA H, TSUBOI A, YAMAGATA H, TSUKAGOSHI N et al. Complete Nucleotide Sequence of a Gene Coding for Heat- and pH-Stable α-Amylase of Bacillus licheniformis: Comparison of the Amino Acid Sequences of Three Bacterial Liquefying α-Amylases Deduced from the DNA Sequences1. The Journal of Biochemistry. 1985;98(5):1147-1156.
8. Wu C, Chung T. Green fluorescent protein is a reliable reporter for screening signal peptides functional in Lactobacillus reuteri. Journal of Microbiological Methods. 2006;67(1):181-186.
9. van Asseldonk M, de Vos W, Simons G. Functional analysis of the Lactococcus lactis usp45 secretion signal in the secretion of a homologous proteinase and a heterologous α-amylase. Molecular and General Genetics MGG. 1993;240(3):428-434.
10. Roos S, Jonsson H. A high-molecular-mass cell-surface protein from Lactobacillus reuteri 1063 adheres to mucus components The GenBank accession number for the sequence reported in this paper is AF120104. Microbiology. 2002;148(2):433-442.
11. Schartmann E( 1 ), Schemmert S( 1 ), Honold D( 1 ), Ziehm T( 1 ), Tusche M( 1 ), Elfgen A( 1 ), et al. In vitro potency and preclinical pharmacokinetic comparison of all-d-enantiomeric peptides developed for the treatment of Alzheimer’s disease. Journal of Alzheimer’s Disease [Internet]. [cited 2021 Oct 9];64(3):859–73. Available from: https://search.ebscohost.com/login.aspx?direct=true&db=edselc&AN=edselc.2-52.0-85049695045&site=eds-live&scope=site