Research - E. coli Auxotrophic Strain Verification of Toshio Iwasaki Group Homepage|Nippon Medical School|日本医科大学|岩崎 俊雄 グループ ホームページ

*We study the structure-function of the intracellular iron-sulfur world in aerobic and thermophilic archaea.LinkIcon

Ec.jpg New Escherichia coli auxotrophic expression strains

EcoliAuxotroph.jpg

This is a collaborative project with Dr. Gennis research group at University of Illinois at Urbana-Champaign, U.S.A., supported in part by the JSPS-NSF International Collaborations in Chemistry Project Grant.

IMPORTANT NOTICE:

(Almost) ALL Escherichia coli auxotrophic expression strains listed in Table 1 are available through the public strain bank, LinkIconAddgene, USA - note that the item "Plasmid" in the table and heading in this link <https://www.addgene.org/Toshio_Iwasaki/> refers to a "Bacterial strain" and not a plasmid (as a result of the default setting of this website), as specified on the individual strain page.
These strains are also available from LinkIconRIKEN Bioresource Center (BRC), Japan <https://dnaconda.riken.jp/search/depositor/dep006963.html>.

Last update: September 26, 2018

PCR Verification of Escherichia coli Auxotrophic Expression Strains (Supporting Information)

Table S1 summarizes the current set of new C43(DE3) and BL21(DE3) auxotrophic expression strains of E. coli designed to facilitate the labeling of either membrane proteins or water-soluble proteins with selected amino acid types enriched with stable isotopes such as 2H, 13C and 15N. The use of a suitable auxotrophic expression strain with the corresponding input isotope labeled amino acid(s) in the growth medium ensures high levels of efficiency as well as selectivity in stable isotope labeling. This page provides the supporting information about these auxotrophic expression strains.

LinkIconBack to the E. coli Auxotrophic Expression Strains Page.

Table S1. New E. coli amino acid auxotrophic host strains used for selective isotope labeling

Strain Precursor strain Genotype
ML2
CLY (Genotype cyo::kan) cyo::kan ilvE
 
ML3 CLY (Genotype cyo::kan) cyo::kan hisG
ML6 ML2 (Genotype cyo::kan) cyo::kan ilvE avtA
ML8 CLY (Genotype cyo::kan) cyo::kan argH
ML12 ML6 (Genotype cyo::kan) cyo::kan ilvE avtA aspC
ML14 C43(DE3) tyrA
ML17 C43(DE3) glnA
ML21 ML14 tyrA hisG
ML24 ML23 cyo ilvE avtA aspC hisG asnA
ML25 ML24 cyo ilvE avtA aspC hisG asnA asnB
ML26 ML23 cyo ilvE avtA aspC hisG argH
ML31 ML26 cyo ilvE avtA aspC hisG argH metA
ML36 ML23 cyo ilvE avtA aspC hisG metA
ML40 ML31 cyo ilvE avtA aspC hisG argH metA lysA
ML41 ML40 cyo ilvE avtA aspC hisG argH metA lysA thrC
ML42 ML41 cyo ilvE avtA aspC hisG argH metA lysA thrC asnB
ML43 ML42 cyo ilvE avtA aspC hisG argH metA lysA thrC asnA asnB
ML45 ML44 cyo ilvE avtA aspC hisG  metA thrC lysA
     
YM138 C43(DE3) cysE
YM154 C43(DE3) cysE  
MS1 YM138 cysE hisG
RF11 C43(DE3) metA
     
RF1 BL21 CodonPlus (DE3)-RIL glyA
RF2 BL21 CodonPlus (DE3)-RIL thrC
RF3  BL21 CodonPlus (DE3)-RIL aspC
RF4 RF3 aspC tyrB
RF5 RF4 aspC tyrB hisG
RF6 BL21 CodonPlus (DE3)-RIL proC
RF8 BL21 CodonPlus (DE3)-RIL asnA asnB 
RF10 BL21 CodonPlus (DE3)-RIL lysA 
RF12 BL21 CodonPlus (DE3)-RIL trpA trpB 
RF13 RF4 aspC tyrB trpA trpB 
RF14 RF13 aspC tyrB trpA trpB serB 
RF15 RF14 aspC tyrB trpA trpB serB glyA 
RF16 RF15 aspC tyrB trpA trpB serB glyA cysE
RF17 RF4 aspC tyrB ilvE
RF18 RF17 aspC tyrB ilvE avtA 
RF21 RF18 aspC tyrB ilvE avtA yfbQ(alaA) yfdZ(alaC)
RF22 RF18 aspC tyrB ilvE avtA asnA asnB
RF23 RF21 aspC tyrB ilvE avtA serB  yfbQ(alaA) yfdZ(alaC)
EH1 RF2 thrC ilvA




Yellow, C43(DE3)-based auxotrophic expression strains. Cyan, BL21(DE3)-based auxotrophic expression strains.
Note that these strains are NOT competent cells and one needs to make them competent before use.

PCR Primers for Verification of Each Target Gene

List of PCR primers used for verification of each knocked-out gene in Table S1 (typical results are shown below).


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Colony PCR Data of Each Auxotrophic Strain in Table S1

Click to magnify each image (and either use browsers "back" button or double click to close the image window).
Left lane, standard marker; right lane, PCR-amplified target gene in the wild-type strain.

ML2.jpg

ML2

ML3.jpg

ML3

ML6.jpg

ML6

ML8.jpg

ML8

ML12.jpg

ML12

ML14v2.jpg

ML14

ML17.jpg

ML17

ML21.jpg

ML21

ML24.jpg

ML24

ML25.jpg

ML25

ML26(S1).jpg

ML26

ML31.jpg

ML31

ML36.jpg

ML36

ML40K1.jpg

ML40K1

ML41 (see below)

ML42.jpg

ML42

ML43.jpg

ML43

ML45 (see below)

YM138.jpg

YM138

YM154.jpg

YM154

MS1.jpg

MS1

RF11.jpg

RF11

RF1.jpg

RF1

RF2.jpg

RF2

RF3(non-auxotroph).jpg

RF3 (non-auxotrophic strain)

RF4.jpg

RF4

RF5.jpg

RF5

RF6.jpg

RF6

RF8.jpg

RF8

RF10.jpg

RF10

RF12.jpg

RF12

RF13.jpg

RF13

RF15.jpg

RF15

RF16.jpg

RF16

RF17.jpg

RF17

RF18.jpg

RF18

RF21.jpg

RF21

RF23 (see below)

EH1.jpg

EH1

Auxotrophic stock strains having a few target genes deleted in an unexpected manner

These stock strains should be used with caution! Colony PCR data (below) indicated that a few target genes (red) were probably deleted in an unexpected manner with the λ-Red recombination system (not tested by direct sequencing), whereas other target genes (black) were knocked out as originally designed. Click to magnify each image (and either use browsers "back" button or double click to close the image window).

ML41(metA?).jpg

ML41 (metA)

ML45(aspC?hisG?).jpg

ML45 (aspC, his G)

RF23.jpg

RF23 (serB, tyrB)

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PCR Primers Used for Construction of the RF Auxotrophic Strains

List of PCR primers used for construction of the RF/YM/EH/MS/ML auxotrophic strains (see Table S1) in steps 1, 2, Fig. S1.


Auxotroph2.jpg

Figure S1. Schematic procedures used for the deletion of a target chromosomal gene with the λ-Red recombination system (steps 1-3). The resistance cassette was removed from the new knock-out strain by FLP recombinase expressed from pCP20 vector (step 4), and a pACYC-based plasmid harboring tRNA genes (argU, ileY, and leuW) for the E. coli rare codons was subsequently incorporated into the resulting cells (step 5) [J. Am. Chem. Soc. 134, 19731-19738 (2012)]. In addition to selective labeling of amino acids, the knockout procedures illustrated here can also be applicable to selectively label biological cofactors (such as hemes, flavins, or ubiquinone) and other metabolites by manipulating the biosynthetic pathways of these compounds. FRT, FLP recombination target.

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Four General Transaminases (or aminotransferases) of Escherichia coli

Generaltransaminases.jpg

Figure S2. Schematic view of selected amino acid biosynthesis pathways in Escherichia coli, catalyzed by four general transaminases (see Fig. 1). They are the products of the ilvE, avtA, aspC and tyrB genes, respectively, and catalyze the interconversion of amino acids and ketoacids by transfer of amino groups, with the overlapping specificities: except for the avtA gene product, the other three general transaminases can use multiple substrates. This poses a major problem for selective labeling with certain amino acids. This scheme can only be used as a general guide with precaution for some amino acids.

For selective labeling with certain amino acids, multiple genetic lesions are required because of the reversible transfer reactions and the overlapping specificities of four general transaminases (or aminotransferases) of E. coli (encoded respectively by ilvE, avtA, aspC and tyrB genes) (Fig. S2). Ideally, the best option in such cases is to use a strain with defects in all four general transaminases (ilvE, avtA, aspC and tyrB).

Thus far, we have been able to obtain such an ideal strain from E. coli BL21(DE3) (Tables 1, S1) but not from C43(DE3) expression host cells, despite multiple attempts using the λ-Red recombination system (Fig. S1). For example, deletion of the ilvE, avtA and aspC genes from the C43(DE3) chromosome resulted in an auxotroph for Ile and Val (Tables 1, 3), and further knockout of the tyrB locus, which was not possible with the C43(DE3) cells for reasons unclear to us, would extend the auxotrophy to include Leu. For Leu auxotrophy of some C43(DE3) auxotroph strains in Table 1, it is therefore necessary to repress the tyrB gene expression by growing the cells in the presence of 0.4-1 mM Tyr in growth medium. 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 genes. It is important to optimize the E. coli expression conditions for each target protein of interest before running selective amino acid isotope labeling experiments.

Similarly, deletion of the both tyrB and aspC genes from the BL21(DE3) chromosome resulted in an auxotroph for Asp (Tables 1, 3), but further knockout of the ilvE locus resulted in a highly complicated auxotroph for Asp, Ile, Leu, Tyr and Phe (to be added; see Fig. S2). Neither the tyrB nor the aspC deletion by itself confers amino acid auxotrophy on the BL21(DE3) cells (Table 1).

Practically speaking, as long as there is a high concentration of amino acids in the growth medium, the collection of the new auxotrophic C43(DE3) and BL21(DE3) expression host strains described here (Table 1) can solve many selective labeling problems, and can be used for cost effective, high-yield production of any recombinant water-soluble or membrane protein that can be expressed in E. coli.

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Primary references to be cited:

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. Pubmed

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. Pubmed

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

  • All ML strains were engineered by Dr. Gennis research group (Myat T. Linn, Robert B. Gennis) at University of Illinois at Urbana-Champaign, U.S.A.
  • YM, MS, EH, and RF strains were engineered by our research group (Yoshiharu Miyajima-Nakano, Risako Fukazawa, Emi Hagiuda, Shinichi Matsushita, Toshio Iwasaki) at Nippon Medical School, Japan, in collaboration with Dr. Gennis research group.

((Almost) ALL these E. coli auxotrophic expression strains listed inTable 1 are available through either LinkIconAddgene, USA (note that the item "Plasmid" in the table and heading in this link<https://www.addgene.org/Toshio_Iwasaki/> refers to a "Bacterial strain" and NOT a plasmid as a result of the default setting of this website, as specified on the individual strain page), LinkIconRIKEN BRC<https://dnaconda.riken.jp/search/depositor/dep006963.html>, Japan, or upon request to T.I. (for all ML, YM, MS, EH, and RF strains listed inTable 1, Nippon Medical School, Japan) or R.B.G. (for all MLstrains only, University of Illinois at Urbana-Champaign, U.S.A.).

  • This strain bank project was supported in part by the International Collaborations in Chemistry Grant from JSPS (T.I.) and NSF (CHE-1026541 to S.A.D.), the JSPS Grant-in-aid 24659202 (T.I.), the Nagese Science and Technology Foundation Research Grant (T.I.), the DE-FG02-87ER13716 (R.B.G.) and DE-FG02-08ER15960 (S.A.D.) Grants from US DOE, NIH & NIGMS Roadmap Initiative (R01GM075937), and NIH grant GM062954 (S.A.D.).

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