Systematic Mutagenesis of E.coli K-12 MG1655 ORFs

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Mutagenesis Project Progress
Construction of Sce-poson mutants
Mutagenesis of Essential Genes
Gene Gorging

Mutagenesis Project (12/12/2003)

An overview of the Systematic Mutagenesis project has been published, as well as papers on Gene Gorging and "Tagalong Mutagenesis".

Number of mutated essential genes: 7
Number of mutant ORFs (Sce-poson mutants): 2001
Total number of strains (+/- pKD46): 3131   [pKD46+: 1830  pKD46-: 1301]

Figure 1. Progress of Systematic Mutagenesis.

Note: The Blattner lab is closed; strains, clones, and vectors from its collection are no longer available. Information about their construction continues to be made available for those who obtained the mutant strains from us in the past.

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Construction of Sce-poson mutants

Y. Kang, T. Durfee, J. D. Glasner, Y. Qiu, D. Frisch, K. M. Winterberg, and F. R. Blattner (2004). "Systematic mutagenesis of the Escherichia coli genome." J Bacteriol 186: 4921-30.

Construction of insertion alleles

We are constructing a set of mutant strains for MG1655. Each mutant strain carries a Tn5 insertion in a specific open reading frame. The following is a general description of the procedures used to construct mutants (see Figure 2). A detailed protocol is available here. To generate these mutants we amplify target genes by PCR and perform an in vitro transposition reaction (Goryshin, I.Y. and Reznikoff, W.S.1998. JBC 273:7367) using one of two modified Tn5-based transposons, EZ-Tn5 <KAN-2> (sequence) or EZ-Tn5 <KAN-I-SceI> (sequence). This reaction creates a set of fragments containing a distribution of independent Tn5 insertions in the ORF sequence. The products of this reaction are electroporated into MG1655 cells expressing the lambda Red recombination proteins from the pKD46 expression plasmid (Datsenko KA, and Wanner BL. 2000. PNAS 97(12):6640). Recombinants are isolated by plating on LB supplemented with kanamycin, to select for the Tn5 insertion allele, and ampicillin to select for maintenance of pKD46.

Confirmation of mutant constructions and determination of the precise locations of Tn5 insertions proceeds through several stages. Initially kanamycin resistant colonies growing at 30 degrees are screened by PCR using primers specific for the targeted locus. The amplification products are analyzed by agarose gel electrophoresis and putative mutants are identified as loci that produce a single band of a size consistent with a Tn5 insertion in the desired ORF. To confirm the correct locus has been targeted, and precisely map the insertion site and orientation within the ORF, the PCR products are then sequenced using a primer that anneals within the Tn5 sequence. It should be noted that the sequences consist of single reads and do not provide accurate coverage of the entire mutant allele.

Figure 2. Strategy for construction of insertion alleles

To cure strains of the temperature sensitive pKD46 plasmid, confirmed mutants are first grown at 43 degrees C. These colonies are streaked on LB plates and an isolated colony is picked and screened for kanamycin resistance and ampicillin sensitivity. We planned to obtain at least one confirmed insertion allele for each ORF, and maintain those strains with and without pKD46.

Replacement of insertions with alternative alleles

To allow the insertion alleles to be efficiently replaced by other types of mutations, we constructed the Tn5<KAN-I-SceI> transposon that contains the recognition sequence for meganuclease I-SceI at one end. There are no naturally occurring I-SceI sites in the E. coli chromosome, and therefore, overexpression of the I-SceI enzyme in the mutant strains creates a lethal double-strand break specifically at the mutated locus. This provides a simple means for counterselecting against the Tn5 insertion by electroporating both an I-SceI expressing plasmid and linear fragments containing the desired allele into the corresponding insertion strain expressing lambda Red (Figure 3). Resultant colonies resistant to the enzyme are expected to be recombinants that have replaced the original Tn5 insertion with the new targeting fragment.

Figure 3. Strategy for replacing insertion alleles with other mutations. An amber allele replacement is shown as an example.

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Mutagenesis of Essential Genes: Conditional lethal mutants

C. D. Herring and F. R. Blattner (2004). "Conditional lethal amber mutations in essential Escherichia coli genes." J Bacteriol 186: 2673-81.

Amber mutations were introduced into essential genes using "Tagalong Mutagenesis". These mutations are conditional lethal based on the use of the arabinose inducible suppressor plasmid pBAD/sup2 (Herring et al. Gene. 2003 Jun 5;311:153-63). They grow normally on RDM plates + Kanamycin + 0.2% L-arabinose, but cannot grow on RDM + Kan + glucose.

7 essential genes were mutated using this method.

strain number gene bnumber product/function
FBSC193 frr b0172 ribosome release factor
FBSC195 gcpE b2515 isoprenoid biosynthesis
FBSC62 lpxC b0096 lipid-A biosynthesis
FBSC197 map b0168 methionine aminopeptidase
FBSC199 murA b3189 peptidoglycan synthesis
FBSC201 ppa b4226 pyrophosphatase
FBSC203 rpsA b0911 ribosomal protein S1

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Gene Gorging

C. D. Herring, J. D. Glasner and F. R. Blattner (2003). "Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli." Gene 311: 153-63.

Gene Gorging allows a researcher to introduce precise sequence modifications into the E. coli genome in a direct way without leaving unwanted drug markers or other "scars." It can be used to make a variety of mutations, such as deletions of changes to the amino acid sequence of a gene. The frequency at which the mutations are introduced is very high; no selection is used - just screening. To introduce a desired mutation into the E. coli genome, gene gorging is carried out using a two-plasmid system shown below (Fig. 4). A donor plasmid contains a PCR product of the mutation-containing fragment, cloned on a standard high copy vector and flanked on one or both ends by the 18 bp restriction site for I-SceI. (Two I-SceI sites work better when making deletions). A mutagenesis plasmid (pACBSR; click here for sequence) carries the I-SceI endonuclease gene and the lambda Red genes under inducible control of the arabinose promoter on a compatible replicon. The donor plasmid is electroporated into E. coli along with the mutagenesis plasmid and plated to select for both plasmids. In this example the plates contain both chloramphenicol and kanamycin. Co-transformants are inoculated into liquid medium containing arabinose to induce expression of I-SceI and Red. The I-SceI meganuclease specifically cuts the donor plasmid, generating many copies of a linear fragment carrying the desired mutation in each cell. The Red gene products facilitate a double recombination at a position on the chromosome homologous to the donor sequence. Clones carrying the desired mutation are identified by screening colonies by PCR or detection of a growth phenotype where feasible.

Fig 4. Strategy of Gene Gorging

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