pBad Expression

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
Or

For 1 liter 10X M9 Salts:

60 g Na2HPO4
30 g KH2PO4
5 g NaCl
10 g NH4Cl
3.373 g Thiamine