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Species: E. coli
Genetic Insert: pstS (A17C,I76G,A197C)
Vector Backbone Description: Vector Backbone:pET22b; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:26199994
Comments: Please note- The numbering system used was for the mature natural wild-type PstS protein. The plasmid produces an extra N-terminal methionine so all mutations will be off by 1nt (e.g., A197C =A198C)
PstS ORF encoding the mature E. coli (A17C, I76G, A197C)PstS protein (PBPw) containing a stop codon at the end of the gene in pET22b (inserted between Nde1 and Xho1 sites in the MCS).
Expression produces MEA at the N-terminus of the mature PstS: So there is an extra M at the N-terminus over the natural, mature PstS. A stop codon was also added at the end of the sequence BEFORE the plasmid-derived His tag. This gives PLY at the C-terminus at the C-terminus of the expressed protein, as in the natural PstS. The amino-acid numbering has been left as based on positions in the natural, mature protein.
Usage
The expressed protein is a weak-binding variant of E. coli phosphate binding protein, PstS. It contains two cysteines (A17C, A197C) and a mutation to weaken binding (I76G). The gene encodes the mature protein without the N-terminal signal peptide, but with an N-terminal methionine. Subsequently the cysteines are labeled with 2 6-iodoactemidotetramethylrhodamines per molecule. This adduct is then used as a phosphate biosensor, having up to 9-fold fluorescence intensity increase on binding inorganic phosphate, with a low dissociation constant so inorganic phosphate can be measured in the tens of micromolar range.
Proper citation: RRID:Addgene_78200 Copy
Species: E. coli
Genetic Insert: parM (K33A D63C T174A T175N D224C C287A)
Vector Backbone Description: Vector Backbone:pHis17; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:20158267
Comments: The plasmid is used to express the plasmid segregation protein, ParM, from E. col. The protein is modified for use as the scaffold of an ADP biosensor, ATR-ParM. Two cysteine mutations are for label attachment (D63C and D224C); there is a mutation to inhibit filament formation (K33A), two mutations to weaken ATP binding (T174A and T175N), a mutation to change the exposed cysteine (C287A) and a C-terminal His6 tag to aid purification. When labelled with two iodoacetamidotetramethylrhodamine (IATR) fluorophores, this biosensor has a fluorescence intensity change of up to 20-fold and can be used to measure ADP in the range of hundreds of nanomolar to ~100 μM, in the presence of up to millimolar ATP. The biosensor can also be used for measurement of other nucleoside diphosphates e.g. GDP, UDP and others.
Proper citation: RRID:Addgene_78202 Copy
Species: E. coli
Genetic Insert: parM (K33A I27C T174A T175N C287A)
Vector Backbone Description: Vector Backbone:pHis17; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:19801632
Comments: The plasmid is used to express the plasmid segregation protein, ParM, from E. col. The protein is modified for use as the scaffold of an ADP biosensor, MDCC-ParM. A single cysteine mutation is for label attachment (I27C); there is a mutation to inhibit filament formation (K33A), two mutations to weaken ATP binding (T174A and T175N), a mutation to change the exposed cysteine (C287A) and a C-terminal His6 tag to aid purification. When labelled with a single N-[2-(1-maleimidyl)ethyl]-7-diethylaminocoumarin-3-carboxamide (MDCC) fluorophore, this biosensor has a fluorescence intensity change of up to 4-fold and can be used to measure ADP in the range of tens of nanomolar to tens of micromolar in the presence of up to~200 μM ATP.
Proper citation: RRID:Addgene_78201 Copy
Species: E. coli
Genetic Insert: pstS (A17C,A197C)
Vector Backbone Description: Vector Backbone:pET22b; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:26199994
Comments: Please note- The numbering system used was for the mature natural wild-type PstS protein. The plasmid produces an extra N-terminal methionine so all mutations will be off by 1nt (e.g., A197C =A198C)
The expressed protein is a double-cysteine variant of E. coli phosphate binding protein. The gene encodes the mature protein without the N-terminal signal peptide, but with an N-terminal methionine, for subsequent labeling at the cysteines with two 6-iodoacetamidotetramethylrhodamines per molecule. This adduct is then used as a fluorescent phosphate biosensor, having 10-15-fold fluorescence increase on binding inorganic phosphate with very high affinity.
Proper citation: RRID:Addgene_78199 Copy
Species: E. coli
Genetic Insert: MetI
Vector Backbone Description: Backbone Marker:Novagen; Backbone Size:5791; Vector Backbone:pET19b; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:22095702
Proper citation: RRID:Addgene_78320 Copy
Species: E. coli
Genetic Insert: MetI
Vector Backbone Description: Backbone Marker:Novagen; Backbone Size:5791; Vector Backbone:pET19b; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:18621668
Proper citation: RRID:Addgene_78316 Copy
Species: E. coli
Genetic Insert: GFPmut2
Vector Backbone Description: Vector Backbone:pZA31-Luc; Vector Types:Bacterial Expression; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:27060147
Proper citation: RRID:Addgene_78493 Copy
Species: E. coli
Genetic Insert: FLIPglu D154A Delta13
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5603; Vector Backbone:pEF/myc/ER; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:20354141
Proper citation: RRID:Addgene_18021 Copy
Species: E. coli
Genetic Insert: FLIPglu F16A Delta13
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5603; Vector Backbone:pEF/myc/ER; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:20354141
Proper citation: RRID:Addgene_18020 Copy
Species: E. coli
Genetic Insert: FLIPglu F16A S112A Delta13
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5428; Vector Backbone:pcDNA3.1(+); Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:20354141
Proper citation: RRID:Addgene_18011 Copy
Species: E. coli
Genetic Insert: FLIPglu D154A Delta13V Venus-Y67G
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5428; Vector Backbone:pcDNA3.1(+); Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:20354141
Proper citation: RRID:Addgene_18016 Copy
Species: E. coli
Genetic Insert: FLIPglu D154A Delta13V ECFP-W67G
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5428; Vector Backbone:pcDNA3.1(+); Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:20354141
Proper citation: RRID:Addgene_18017 Copy
Species: E. coli
Genetic Insert: FLIPglu F16F Delta13
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5428; Vector Backbone:pcDNA3.1(+); Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:20354141
Proper citation: RRID:Addgene_18018 Copy
Species: E. coli
Genetic Insert: FLIPglu F16A S112A Delta13
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5603; Vector Backbone:pEF/myc/ER; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:20354141
Proper citation: RRID:Addgene_18019 Copy
Species: E. coli
Genetic Insert: LetB (a.k.a. b1834, yebT, ECK1833)
Vector Backbone Description: Backbone Marker:Novagen; Backbone Size:5523; Vector Backbone:pET21a; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:35077766
Proper citation: RRID:Addgene_181817 Copy
Species: E. coli
Genetic Insert: LetB (a.k.a. b1834, yebT, ECK1833)
Vector Backbone Description: Backbone Marker:Novagen; Backbone Size:5523; Vector Backbone:pET21a; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:35077766
Proper citation: RRID:Addgene_181816 Copy
Species: E. coli
Genetic Insert: LetB (a.k.a. b1834, yebT, ECK1833)
Vector Backbone Description: Backbone Marker:Novagen; Backbone Size:5523; Vector Backbone:pET21a; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:35077766
Proper citation: RRID:Addgene_181815 Copy
Species: E. coli
Genetic Insert: EcoRI endonuclease & EcoRI methylase
Vector Backbone Description: Backbone Marker:n/a; Backbone Size:3000; Vector Backbone:pSCC3; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:2745418
Comments: pSCC3 plasmid derivation is described in King, K. et al. (1989) J.Biol.Chem. 264, 11807-11815.
Please note that this plasmid may require a unique bacterial strain, so make sure to confirm that you can also obtain the appropriate growth strain. Please contact us at [email protected] or contact our distributors if you have any questions.
Proper citation: RRID:Addgene_40190 Copy
Species: E. Coli
Genetic Insert: Bistable switch using dynamic degradation
Vector Backbone Description: Backbone Size:3460; Vector Backbone:psc101; Vector Types:Bacterial Expression, Synthetic Biology; Bacterial Resistance:Kanamycin
Defining Citation: PMID:22776405
Proper citation: RRID:Addgene_40108 Copy
Species: E. Coli
Genetic Insert: CI inducible sfGFP
Vector Backbone Description: Backbone Size:2097; Vector Backbone:p15A; Vector Types:Bacterial Expression, Synthetic Biology; Bacterial Resistance:Ampicillin
Defining Citation: PMID:22776405
Proper citation: RRID:Addgene_40127 Copy
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