C-DAG Amyloidogenicity Kit

Curli-dependent amyloid generator (C-DAG) Amyloidogenicity Kit (VS45 Strain, pVS72, and pVS105) is a bacteria-based methodology for the rapid generation of amyloid aggregates of a given amyloidogenic protein.

Highlights

  • Simple method for identifying amyloidogenic proteins and for distinguishing between amyloidogenic and non-amyloidgenic variants of a particular protein
  • Assay for amyloid aggregation without the need for protein purification
  • Compatible with multiple downstream detection methods (e.g., Colony screening using amyloid-binding Congo red (CR) dye, transmission electron microscopy (TEM), SDS-stability assays, etc.)

Kit Components

  • E. coli strain VS45 used for the export of fusion proteins via the curli secretion pathway
  • pExport plasmids: bla PBAD csgAss-X-His6, pBR322 ori; produces CsgA residues 1 – 42 fused to a test protein, X, with a C-terminal His6-tag. Where X = sup35[NM] in plasmid pVS72, and sup35[M] in plasmid pVS105

The curli-dependent amyloid generator (C-DAG) methodology takes advantage of the natural ability of E. coli cells to elaborate surface associated amyloid fibers known as curli. The major curli fiber sub-unit is the amyloidogenic protein CsgA, which is secreted from the cell under the control of an N-terminal signal sequence. The transfer of this signal sequence to the N-terminus of heterologous amyloidogenic proteins similarly directs their export to the cell surface, where they assemble as amyloid fibrils. Importantly, protein secretion through the curli export pathway facilitates acquisition of the amyloid fold specifically for proteins that have an inherent amyloid forming propensity.

From the laboratory of Ann Hochschild, PhD, Harvard University.

The Investigator's Annexe Part of The Investigator's Annexe program.

Catalog Number Product Size AVAILABILITY Price Qty
EF2001
C-DAG Amyloidogenicity Kit (VS45 Strain, pVS72, and pVS105)
Kit (1 of each) In stock
Price: $646.00
EF2002
E. coli Exporter Strain VS45, 2x50uL
2x50uL (frozen) In stock
Price: $321.00
EF2003
pExport (pVS72) Positive Control Plasmid, 250ng
250ng (liquid) In stock
Price: $213.00
EF2004
pExport (pVS105) Negative Control Plasmid, 250ng
250ng (liquid) In stock
Price: $213.00
Specifications
Product Type: Kit
Components: One each of EF2002, EF2003, and EF2004
Documentation

PDF C-DAG Amyloidogenicity Kit Protocol


E. coli Exporter Strain VS45 (Cat. EF2002)

Organism: Escherichia coli
Genotype: [derivative of strain MC4100] F-, [araD139]B/r, Δ(argF-lac)169, λ-, e14-, flhD5301, Δ(fruK-yeiR)725(fruA25), relA1, rpsL150(strR), rbsR22, Δ(fimB-fimE)632(::IS1), deoC1, Δ(csgBAC) [containing plasmid pVS76; cat, PlacUV5-csgG, pACYC184 ori; produces CsgG]
Cell Line Name: VS45
Competency: MES (2-[N-morpholine]ethanesulfonic acid) chemical competent cells
Antibiotic Resistance: Streptomycin (encoded on chromosome), chloramphenicol (encoded on plasmid pVS76)
Growth Medium: LB liquid or agar media, supplemented with the appropriate antibiotics, at 37˚C. (See attached PDF for more information)
Induction: To induce the expression of csgG from plasmid pVS76, the growth medium is supplemented with 1 mM IPTG. To induce the expression of the fusion gene for export from the pExport plasmid, the growth medium is supplemented with 0.2% arabinose.
Storage Temperature: -80C
Shipped: Dry Ice

pExport (pVS72) Positive Control Plasmid (Cat. EF2003)

Gene Insert Name: pVS72: sup35[NM], encoding residues 1 – 253
Insert Size: pVS72: 759 bp (sup35[NM])
Species: sup35 coding sequence is of S. cerevisiae origin. The pExport plasmid is a derivative of a commonly used plasmid vector for cloning and protein expression in E. coli
Fusion Proteins/Tags: pVS72 encodes CsgAss-(Ala)3-Sup35[NM]-(His)6, where CsgAss is the CsgA secretion signal
Vector Backbone and Size (bp): pBR322 derivative: 5434 bp for pVS72 
Cloning site 5' : NotI (between csgAss and sup35[NM]) 
Cloning site 3' : XbaI (immediately after the STOP codon) 
Antibiotic Resistance: Ampicillin  
High or Low Copy: High
Grow in Standard E. coli @ 37C?: Yes
Selectable Markers: None
Storage Temperature: -20C
Shipped: Room Temperature

pExport (pVS105) Negative Control (Cat. EF2004)

Gene Insert Name: pVS105: sup35[M], encoding residues 125 – 253
Insert Size: pVS105: 387 bp (sup35[M])
Species: sup35 coding sequence is of S. cerevisiae origin. The pExport plasmid is a derivative of a commonly used plasmid vector for cloning and protein expression in E. coli
Fusion Proteins/Tags: pVS105 encodes CsgAss-(Ala)3-Sup35[M]-(His)6, where CsgAss is the CsgA secretion signal
Vector Backbone and Size (bp): pBR322 derivative: 5062 bp for pVS105
Cloning site 5' : NotI (between csgAss and sup35[M]) 
Cloning site 3' : XbaI (immediately after the STOP codon) 
Antibiotic Resistance: Ampicillin
High or Low Copy: High
Grow in Standard E. coli @ 37C?: Yes
Selectable Markers: None
Storage Temperature: -20C
Shipped: Room Temperature
Data
Plasmid Map & Data
Plasmid Map and Data

(LEFT) pExport plasmid map with relevant features. In the example above ,the pExport plasmid, pVS72, contains Sup35 NM as the gene of interest. All restriction sites shown are unique. (RIGHT) Results for Sup35 NM and Sup35 M when using C-DAG and tested in various assays. (A) When grown on solid medium supplemented with CR, E. coli cells producing CsgAss-NM form colonies that stain red, whereas cells producing CsgAss-M form pale colonies (reprinted, with permission, from ref. 5). (B) E. coli cells secreting CsgAss-NM produce fibrillar aggregates that can be visualized by transmission electron microscopy (TEM), whereas cells secreting CsgAss-M do not produce fibrillar aggregates. (C) The fibrillar aggregates generated by cells secreting CsgAss-NM are immunolabeled by a Sup35 antibody. No fibrillar aggregates are detected for cells secreting CsgAss-M (reprinted, with permission from ref. 5). (D) E. coli cells secreting CsgAss-NM produce material that manifests apple-green birefringence when viewed by bright-field microscopy between crossed polarizers, whereas cells secreting CsgAss-M do not. Cell samples are taken from colonies formed on solid medium supplemented with Congo Red. (E) SDS-resistant aggregates are detected using the filter-retention assay for samples of cells secreting CsgAss-NM, but not for cell samples secreting CsgAss-M.

Adapted from: Sivanathan V. & Hochschild A., Nat Prot. (2013) 8:1381-1390.

Provider
From the laboratory of Ann Hochschild, PhD, Harvard University.
Comments
Plasmid Notes

To replace sup35[NM] or sup35[M] with your gene of interest, PCR amplify your gene of interest using primers that provide the NotI and XbaI restriction sites, as well as the (His)6 tag and STOP codon, as described below and in ref(1):

Forward primer:
5' TATA GCG GCC GCA xxx ...., with xxx encoding the first codon of your gene of interest
GCG GCC GCA encodes the NotI restriction site as well as a (Ala)3 linker between the csgAss and your protein of interest

Reverse primer: (listed as the reverse complement of the coding strand)
5' TATA TCTAGA TTA gtgatgatggtgatggtg yyy ...., with yyy encoding the last codon of your gene of interest
TCTAGA encodes the XbaI restriction site
TTA encodes the STOP codon
gtgatgatggtgatggtg encodes (His)6

References
  1. Sivanathan, V & Hochschild A., (2013) A bacterial export system for generating extracellular amyloid aggregates. Nat Prot. 8:1381-1390
  2. Sivanathan, V & Hochschild A., (2012) Generating extracellular amyloid aggregates using E. coli cells. Genes Dev 26:2659-2667

If you publish research with this product, please let us know so we can cite your paper.

 
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