Explaining our choices of chaperone systems and substrate proteins in our project
Explaining the choice of GroES
The GroES chaperone was chosen because it has not yet been studied individually in vivo and may have an holdase activity [1]. Its ability to dynamically remodel substrate proteins with and without GroEL, in relevant concentrations [1].
Explaining the choice of backbone
Our biobrick was placed in the pSB4A5 backbone to ensure that the biobrick could be co-expressed with the chaperone plasmid pGreE7 from Takara and substrate proteins mNG-Aβ1-42, Tau0N4R-EGFP, EGFP-Aβ1-42 and α-synuclein-EGFP. This is due to all of them having different classes of origin of replication (ORI) [2, 3]. It also had a selectable marker (AmpR) that didn’t exist in any of the other plasmids.
E.coli (BL21) prepared with Takara plasmids in different combinations was generously provided by IFM-Chemistry at Linköping University. The biobrick in the pSB4A5 backbone was transformed together with the substrate protein into E.coli (BL21) together with the Takara plasmid, and selected through triple antibiotic selection (KanR, AmpR, CmR).
Explaining choice of promoters and substrate proteins
Having three different promoters with different inducers (IPTG, L-arabinose, tetracycline) enables variation of expression. This ensures that multiple parameters can be changed. Variation of expression allows us to change chaperone expression to find optimal conditions for the substrate protein of choice. The substrates were chosen because of their difficult fold-process (all fusion proteins). The result of the integrated human practice questionnaire contributed to this as well. More information on why the substrate proteins were chosen can be found under integrated human practice.
Aβ1-42, Tau0N4R, α-synuclein linked with EGFP or mNeonGreen(mNG) were also chosen for its aggregation-prone behaviour [4,5,6]. The flourescent protein acted as reporters for protein expression. mNG was originally created by Shaner NC et. al and is one of the brightest fluorescent proteins and a suitable tag for fusion proteins [7]. Using the T7-polymerase for the substrate will ensure a strong expression [3].
Explaining combination of plasmids
We also had a plasmid with the GroE system (Takara) which we tried as well. This was the most relevant chaperone-system to study in concert with our plasmid. The even-higher molar ratio of GroES:GroEL in the GroE system should further increase the efficiency of this system [1].
All systems were also expressed without the GroE-system, and the GroE-system was expressed without our biobrick chaperone. This was done to control the need of overexpression of any system.
Plasmids used in our project
Name
Marker
Class
Promoter
Insert
pGroE7
Chloramphenicol
B
pBAD
GroE
pmNG-Aβ
Kanamycin
A
pT7
mNG-Aβ
pTau-EGFP
Kanamycin
A
pT7
Tau-EGFP
pEGFP-Aβ
Kanamycin
A
pT7
EGFP-Aβ
pSynEGFP
Kanamycin
A
pT7
α-synuclein-EGFP
pSB4A5-GroES
Ampicillin
C
pTet
GroES
[1] Moparthi SB, Sjölander D, Villebeck L, Jonsson BH, Hammarström P, Carlsson U. Transient conformational remodeling of folding proteins by GroES - Individually and in concert with GroEL. J Chem Biol. 2014;7(1):1–15.
[2] Chaperone Plasmid Set For Research Use v201701Da [Internet]. [cited 2018 Aug 12]. Available from: http://www.takara-bio.com.
[3] Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol. 2014;5:172.
[4] Fitzpatrick AWP, Falcon B, He S, Murzin AG, Murshudov G, Garringer HJ, et al. Cryo-EM structures of tau filaments from Alzheimer’s disease. Nature [Internet]. 2017;547(766).
[5] BioLegend. Anti-beta-amyloid, 1-16 antibody. [Internet]. Available from: https://www.biolegend.com/en-us/products/anti-beta-amyloid--1-16-antibody-10998
[6] Breydo L. Wu W. J. Uversky V. α-Synuclein misfolding and Parkinson´s disease. [Internet]. Biochim. Biophys. Acta, 1822 (2012), pp. 261-285
[7] Shaner NC, Lambert GG, Chammas A, Ni Y, Cranfill PJ, Baird MA, et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods [Internet]. 2013;10(5):407–9. Available from: http://www.nature.com/doifinder/10.1038/nmeth.2413
