Bacterial strains and plasmids
Bacterial strains and deoxynucleic acids (DNAs) used in this study are
listed Table 1. Genomic DNA from bacterial cells was isolated using the
Blood & Tissue DNA Isolation Kit (Qiagen, Hilden, Germany). Standard
recombinant DNA technologies were applied for molecular cloning
(Miller 1992). Taq DNA polymerase
was obtained from New England Biolabs (Ipswich, MA, USA). All
synthesized oligonucleotides were obtained from Integrated DNA
Technologies (Coralville, IA, USA). DNA sequencing was conducted by the
Centre for Applied Genomics at the Hospital for Sick Children (Toronto,
Canada). E. coli BW25113 was the parental strain for derivation
of all engineered strains in this study and E. coli DH5α was used
as a host for molecular cloning. Note that the ldhA gene
(encoding lactate dehydrogenase) was previously inactivated in BW25113,
generating BW∆ldhA (Srirangan et
al. 2014).
For genetic implementation of the Shemin pathway in E. coli , thehemA gene was first amplified by polymerase chain reaction (PCR)
using the primer set g-hemA and the genomic DNA of wild-typeRhodobacter sphaeroides DSM 158 as the template. The amplifiedhemA gene was Gibson–assembled with the PCR-linearized pK184
using the primer set g-pK-hemA to generate pK-hemA. The expression of
the cloned hemA gene in the pK184 vector was under the control of
the Plac promoter.
Knockouts of the genes, including sdhA (encoding succinate
dehydrogenase complex flavoprotein subunit A, SdhA) and iclR(encoding transcriptional AceBAK operon repressor, IclR), were
introduced into BW∆ldhA by P1 phage transduction
(Miller 1992) using the appropriate Keio
Collection strains (The Coli Genetic Stock Center, Yale University, New
Haven, CT, USA) as donors (Baba et al.
2006). To eliminate the co-transduced FRT-KnR-FRT
cassette, the transductants were transformed with pCP20
(Cherepanov and Wackernagel 1995), a
temperature sensitive plasmid expressing a flippase (Flp) recombinase.
Upon Flp-mediated excision of the KnR cassette, a
single Flp recognition site (FRT “scar site”) was generated. Plasmid
pCP20 was then cured by growing cells at 42°C. The genotypes of derived
knockout strains were confirmed by colony PCR using the appropriate
verification primer sets listed in Table 1.
The expression of hemB was repressed by CRISPRi using various
derived plasmids from pdcas9-bacteria (Addgene plasmid # 44249) and
pgRNA-bacteria (Addgene plasmid # 44251). All synthesized
oligonucleotide pairs have 60 nucleotides (nt), which includes 20 nthemB -targeting sequence, 20 nt upstream and 20 nt downstream
sequences of pgRNA-bacteria vector (Fig. 2). They were ordered from
Integrated DNA Technologies (Coralville, IA, USA) and annealed as
described previously (Pengpumkiat et al.
2016), generating four double-stranded DNA fragments ofhemB -gRNA-L1, hemB -gRNA-L2, hemB -gRNA-L3, andhemB -gRNA-L4 (Table 1). All these four DNA fragments were
individually Gibson-assembled with the PCR-linearized pgRNA-bacteria
using the primer set g-pgRNA to generate pgRNA-L1, pgRNA-L2, pgRNA-L3,
and pgRNA-L4, respectively (Table 1). The four hemB -repressed
strains, i.e., DMH-L1, DMH-L2, DMH-L3, and DMH-L4, were developed by
creating a triple-plasmid system (Fig. 2) containing pK-hemA,
pdcas9-bacteria, and the gRNA-containing plasmid (i.e., pgRNA-L1,
pgRNA-L2, pgRNA-L3, or pgRNA-L4). For the control strain DMH-CT, the
original pgRNA-bacteria plasmid without any hemB -targeting
sequence was used as the third plasmid.