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.
Table S1. New E. coli amino acid auxotrophic host strains used for selective isotope labeling
||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|
|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|
|RF1||BL21 CodonPlus (DE3)-RIL||glyA|
|RF2||BL21 CodonPlus (DE3)-RIL||thrC|
|RF3||BL21 CodonPlus (DE3)-RIL||aspC|
|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)|
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.
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.
Auxotrophic stock strains having a few target genes deleted in an unexpected manner
The following 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).
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.
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.
Four General Transaminases (or aminotransferases) of Escherichia coli
Figure S2. Schematic view of selected amino acid biosynthesis pathways in Escherichia coli, catalyzed by four general transaminases (see Fig. 1 [J. Biochem. (Review) 169, 387-394 (2021)]). 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). In principle, the potential use of strains with defects in all four general transaminase genes (ilvE, avtA, aspC and tyrB) of E. coli should help minimize possible dilution and scrambling of the input amino acid label whenever available (J. Biomol. NMR 8, 184-192 (1996)) - in practice, some of these strains appear to exhibit complicated patterns of amino acid combination requirements for growth or they grow very slowly (J. Biochem. (Review) 169, 387-394 (2021)), so careful planning of experimental conditions is required (Tables 1, S1):
- (i) neither the tyrB nor the aspC deletion by itself confers amino acid auxotrophy on the BL21(DE3) cells (Table 1);
- (ii) strains having knockouts in both aspC and tyrB genes (RF4, RF5, RF13, RF14, RF15, RF16, RF17, RF18 and RF21) require Asp (but not Glu) for growth in M63 minimal media, and some of them (RF15, RF16, RF17, RF18 and RF21) grow only slowly in M63 minimal media in the presence of the L-amino acids specified in Table 1;
- (iii) strain RF17, having knockouts in the aspC, tyrB and ilvE genes, requires the presence of Asp, Tyr, Phe, Ile and Leu for (slow) growth in M63 minimal media;
- (iv) strains RF18 and RF21, having knockouts in all four of the general transaminase genes (aspC, tyrB, ilvE and avtA ), appear to require the presence of Asp, Tyr, Phe, Ile, Leu and Val for slow growth in M63 minimal media - of these, although strain RF21 has further knockouts in genes yfbQ (alaA) and yfdZ (alaC), it requires the presence of Asp, Tyr, Phe, Ile, Leu and Val for slow growth in M63 minimal media like RF18, and is not an Ala auxotroph either [cf., RF16, having knockouts in aspC, tyrB, trpA, trpB, glyA, serB and cysE genes and representing an ideal genotype to selectively label Ser, unexpectedly represents a new BL21(DE3) derived Ala auxotroph, which requires the presence of Asp, Tyr, Trp, Gly, Ser, Cys and Ala for very slow growth in M63 minimal medium, but does not grow in M63 minimal medium in the presence of Asp, Tyr, Trp, Gly, Ser and Cys like RF15] (J. Biochem. (Review) 169, 387-394 (2021));
- (v) considering also the regulation of the Asp degradation pathways by feedback inhibition by Thr and Lys, and some form of repression by Thr, Ile, Lys and Met, further deletions of asnA and asnB in RF18 would result in a BL21(DE3) derivative requiring the presence of Asp, Tyr, Phe, Ile, Leu and Asn for growth in M63 minimal media, which is expected to be applicable to selective labelling of Asp at least for short-term cultivations grown in medium supplied with sufficient amounts of Thr, Ile, Lys and Met (J. Biochem. (Review) 169, 387-394 (2021)).
Thus far, we have been able to obtain such "ideal" strains from E. coli BL21(DE3) (RF18 and RF21 in Tables 1, S1 (J. Biochem. (Review) 169, 387-394 (2021))) 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 (J. Biochem. (Review) 169, 387-394 (2021)). 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.
The complicated patterns of amino acid requirements for bacterial growth in these strains certainly reflect the significant overlap in cognate biosynthetic and biodegradation pathways (see Fig. 1), and our knowledge about the complicated regulation of the metabolic flow is still incomplete. It is therefore important to optimize the E. coli expression conditions for each target protein of interest before running selective amino acid isotope labeling experiments.
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 (Tables 1, S1) 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.
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
Iwasaki, T.,* Miyajima-Nakano, Y., Fukazawa, R., Lin, M. T., Matsushita, S., Hagiuda, E., Taguchi, A. T., Dikanov, S. A., Oishi, Y., and Gennis, R. B. (2021) Escherichia coli amino acid auxotrophic expression host strains for investigating protein structure-function relationships. J. Biochem. (Review), 169, 387-394. 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, Shin-ichi Matsushita, Toshio Iwasaki) at Nippon Medical School, Japan, in collaboration with Dr. Gennis research group.
(Almost) ALL these E. coli auxotrophic expression strains listed in Table 1 are available through either Addgene, 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), RIKEN BRC, Japan <https://dnaconda.riken.jp/search/depositor/dep006963.html>, or upon request to T.I. (for all ML, YM, MS, EH, and RF strains listed in Table 1, Nippon Medical School, Japan) or R.B.G. (for all ML strains 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.).