RF21
(Bacterial strain #102803)

• Purpose: BL21(DE3)-based E. coli amino acid auxotrophic host strain used for selective isotope labeling. aspC tyrB ilvE avtA yfbQ(alaA) yfdZ(alaC) genes deleted. (yfbQ also called alaA; yfdZ also called alaC)
• Depositing Labs: Robert Gennis, Toshio Iwasaki
• Publication: Iwasaki et al J Biochem. 2021 Apr 29;169(4):387-394. doi: 10.1093/jb/mvaa140.

Backbone

• Vector backbone: none

Growth in Bacteria

• Bacterial Resistance(s): None
• Growth Temperature: 37°C
• Growth Strain(s): RF21 (aspC tyrB ilvE avtA yfbQ(alaA) yfdZ(alaC))
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: none

Resource Information

• Supplemental Documents:
  • PCR Data RF21
  • PCR Primers

Terms and Licenses

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

Depositor Comments

This work is supported in part by JSPS-NSF International Collaborations in Chemistry (ICC) research grant.

Genotype= aspC tyrB ilvE avtA yfbQ(alaA) yfdZ(alaC)
Precursor strain = RF18
Modified from the parent Escherichia coli BL21(DE3) strain

Selective amino acid labeling (and/or requirement) = Asp, Tyr, Phe, Ile, Leu, Val####

#### RF21 has knockouts in the four general transaminase genes of E. coli (aspC, tyrB, ilvE, and avtA) and is found to require the presence of L-Asp, L-Tyr, L-Phe, L-Ile, L-Leu plus L-Val for slow growth in M63 minimal medium. Although RF21 strain has further knockouts in yfbQ (alaA) and yfdZ (alaC) genes, it is NOT an L-Ala auxotroph, either (requiring the presence of L-Asp, L-Tyr, L-Phe, L-Ile, L-Leu plus L-Val for slow growth in M63 minimal medium, like RF18).

Please visit the following links for additional details on this strain and selective amino acid labeling-
http://www2.nms.ac.jp/fesworld/EcoliStrains.html
http://www2.nms.ac.jp/fesworld/EcoliStrainsSuppl.html

Note that these strains are NOT competent cells and one needs to make them competent before use.

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.

Supporting References:
Lin, M. T., Fukazawa, R., Miyajima-Nakano, Y., Matsushita, S., Choi, S. K., Iwasaki, T., and Gennis, R. B. (2015) Escherichia coliauxotroph host strains for amino acid-selective isotope labeling of recombinant proteins. Methods Enzymol. (Isotope Labeling of Biomolecules - Labeling Methods), 565, 45-66.

Iwasaki, T., Fukazawa, R., Miyajima-Nakano, Y., Baldansuren, A., Matsushita, S., Lin, M. T., Gennis, R. B., Hasegawa, K., Kumasaka, T., and Dikanov, S. A. (2012) Dissection of hydrogen bond interaction network around an iron-sulfur cluster by site-specific isotope labeling of hyperthermophilic archaeal Rieske-type ferredoxin. J. Am. Chem. Soc. 134, 19731-19738.

Lin, M. T., Sperling, L. J., Frericks Schmidt, H. L., Tang, M., Samoilova, R. I., Kumasaka, T., Iwasaki, T., Dikanov, S. A., Rienstra, C. M., and Gennis, R. B. (2011) A rapid and robust method for selective isotope labeling of proteins. Methods 55, 370-378.