ML12
(Bacterial strain #61910)

• Purpose: C43(DE3)-based E. coli amino acid auxotrophic host strains used for selective isotope labeling. ilvE avtA aspC genes deleted.
• Depositing Labs: Robert Gennis, Toshio Iwasaki
• Publication: Lin et al Methods. 2011 Dec;55(4):370-8. doi: 10.1016/j.ymeth.2011.08.019. Epub 2011 Sep 8.

Backbone

• Vector backbone: none

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): ML12 (cyo::kan ilvE avtA aspC)
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: none

Resource Information

• Supplemental Documents:
  • PCR primers
  • PCR data ML12

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Genotype = cyo::kan ilvE avtA aspC (Kan cassette in the chromosomal DNA)
Precursor strain = ML6 (Genotype cyo::kan)
Selective amino acid labeling (and/or requirement) = (Ala#), Ile, Leu*, Tyr#, Val

CLY strain was derived from the C43(DE3)strain by transferring the cyo deletion (with kanamycin resistance cassette kanR) from an E. coli B strain to C43(DE3) strain by phage P1 [J. Biol. Chem. 282, 8777-8785 (2007)]. Thus, the kanR casette CANNOT be removed because there are NO FRT sites flanking kanR.

*In the presence of 0.4-1 mM Tyr, tyrB is repressed and Leu is required for growth in minimal medium. This strategy can only be applicable for a short-term cultivation but not suitable for a long-term cultivation for heterologous expression of foreign genes [Methods 55, 370-378 (2011)].

#In the presence of 0.4-1 mM Tyr, tyrB is repressed and Tyr is required for growth in minimal medium. Under these conditions, this strain can also be used for selective labeling of the input Tyr and/or Ala label(s), although minor diffusion of the input label(s) can occur (see Tables 2, 3). Note that this strategy can only be applicable for a short-term cultivation but not suitable for a long-term cultivation for heterologous expression of foreign geness [Methods 55, 370-378 (2011)].

Each target gene was deleted from the chromosome of C43 (DE3) E. coli strain using λ-Red recombination system.

Supplemental documents contain a list of PCR primers used for verification of each knocked-out gene as well as an image showing PCR results for this strain.