L-arabinose: 150.1 g/mol (Sigma, catalog no. A3256)

Best conditions for induction with arabinose: Induced expression levels are generally higher in minimal-defined medium than in rich medium. This is due to trace amounts of sugars (i.e. glucose) in rich medium that lower expression levels  slightly through the catabolite repression pathway. The pBAD Expression System contains the prototrophic E. coli strain LMG194 that grows well in minimal-defined medium to maximize the yields of the expressed protein.

Induction of the pBAD promoter when cells are growing in LB or RM-Glucose: If the construct is grown under maximal repression, i.e., in LMG194 with D-glucose in RM media, the culture must be spun down and resuspended in RM containing glycerol and Arabinose.

Addendum: Use 0.2% glycerol in the RM medium (basically substitute glycerol for the glucose in the media recipe)

When using TOP10 and growing them in LB, since they will not grow in RM, Arabinose can be added directly to the culture.

Benefit of arabinose induction vs. IPTG induction: Arabinose induction is titratable: the higher the concentration of Arabinose, the stronger the expression. This allows for expression to be fine-tuned which may be important if the protein has toxicity or solubility problems. Arabinose is also less expensive than IPTG.

For cloning and expression, a recA, endA strain is recommended; such as TOP10. 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 other strains with araBADC- and araEFGH+ may be suitable for general use.

The E. coli strain LMG194 (Guzman et al., 1995) allows 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-

Genotype of TOP10
F- mcrA.(mrr-hsdRMS-mcrBC) Δ80lacZ M15.lacX74 recA1 araD139.(araA-leu)7697 galU galK rpsL endA1 nupG.

Note: 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.

Genotype of LMG194
F-.lacX74 gal E thi rpsL (StrR).phoA (Pvu II).ara714 leu::Tn10.
Note: This strain is deleted for araBADC. It is also streptomycin and tetracycline resistant.

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 promoter, 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

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: Manufacturing plates the LMG194 strain on LB+Tet plates that contain 25 mg/mL tetracycline. Streptomycin is not used for selection.