Paul.xu at 14:28, 27 December 2018

2018-12-27T14:28:34Z

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For cloning and expression, a '''recA, endA '''strain is recommended; such as TOP10 (included in the kit). This strain is capable of transporting L-arabinose, but not metabolizing it. This is important for expression studies as the level of L-arabinose will be constant inside the cell and not decrease over time. Note that  3other strains may be suitable for general use. Be sure to check the genotype of your strain. It should be '''araBADC'''- and '''araEFGH +.'''

'''TOP10:''' F- mcrA Δ( mrr-hsdRMS-mcrBC) Φ80lacZΔM15 Δ lacX74 '''recA1''' araD139 Δ( araleu)7697 galU galK rpsL (StrR) '''endA1 '''nupG .This strain is araBADC– . It is deleted for both araBA and araC, and the gene for araD has a point mutation in it, making it inactive. 

'''Nico21(DE3):  '''can::CBD fhuA2 [lon] ompT gal (λ DE3) [dcm] arnA::CBD slyD::CBD glmS6Ala ∆hsdS λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5, Deficient in proteases Lon and OmpT,Resistant to phage T1 (fhuA2)

'''NEB 10-beta/Stable''': Δ(ara-leu) 7697 '''araD139''' fhuA ΔlacX74 galK16 galE15 e14- ϕ80dlacZΔM15 '''recA1''' relA1 '''endA1''' nupG rpsL (StrR) rph spoT1 Δ(mrr-hsdRMS-mcrBC), Reduced recombination of cloned DNA (recA1), Activity of nonspecific endonuclease I (endA1) eliminated for highest quality plasmid preparations, Resistance to phage T1 (fhuA2)

'''NEBTurbo:''' F' proA+B+ lacIq ∆lacZM15 / fhuA2  ∆(lac-proAB)  glnV galK16 galE15  R(zgb-210::Tn10)TetS  '''endA1''' thi-1 ∆(hsdS-mcrB)5  ,  K12 Strain, Activity nonspecific endonuclease I (endA1) eliminated,  Resistance to phage T1 (fhuA2)

The E. coli strain '''LMG194''' (Guzman et al., 1995) is included in the kit to allow additional repression for low basal level expression of toxic genes. This strain is capable of growth on minimal medium (RM medium), which allows repression of pBAD by glucose. The LMG 194 strain can be purchased from ATCC (ATCC # 47090).

Other E. coli strains that can be used for pBAD expression: Generally any E. coli strain that is '''Ara- '''could be used for expression. Some common genotypes: TOP10 cells are Ara- TOP10F' cells are Ara- '''DH5alpha are not Ara-''' '''INValphaF' cells are not Ara-'''
OmniMAX, Mach1, and BL21(DE3) are not araBADC-.

Genotype of '''LMG194: '''F-.lacX74 gal E thi rpsL.phoA (Pvu II).ara714 leu::Tn10.  this strain is streptomycin and tetracycline resistant. 

Note: This strain is deleted for araBADC. It is also streptomycin and tetracycline resistant.

For toxic protein use '''LMG194.'''

'''Advantage of LMG194 over TOP10''': LMG194 is not absolutely necessary since the TOP10 strain may also be used for expression. However, the LMG194 strain is provided to allow for growth of the construct under conditions of maximal  repression. LMG194 can grow on minimal media allowing for utilization of specific carbon sources. '''Glucose is the sole carbon source that represses the pBAD promote'''r,'''while Glycerol does not repress'''. TOP10 cannot be grown in minimal media and other components in LB, such as sugars and other catabolites, may interfere with the gene regulation of arabinose.

 Media to grow LMG194: '''LMG194 will grow in LB, RM, but not M9'''. RM is used to ensure low basal level expression from the pBAD promoter.

 Thiamine and leucine: The '''LMG194 genotype requires thiamine and leucine''' for growth. The leucine is provided in the Casamino acids. Thiamine should be included when making M9 medium.

To make 1 L of 1X M9 medium, add 1 mL of 1 M thiamine Or For 1 liter 10X M9 Salts: 60 g Na2HPO4 30 g KH2PO4 5 g NaCl 10 g NH4Cl 3.373 g Thiamine

 LacZ gene in LMG194: LMG194 cells carry the lacX74 mutation. This mutation deletes the entire lac operon from the chromosome. Therefore, the lacZ gene is not present in LMG194 cells. Antibiotics used for selecting the LMG194 strain: Make plates the LMG194 strain on'''LB+Tet''' plates that contain 25 mg/mL tetracycline. Streptomycin is not used for selection.
[[Category:Protocols]]

Arabinose Induction - Revision history

Paul.xu at 14:28, 27 December 2018