Supplementary Methods
Fragments for thrBC disruption
DNA fragments containing upstream and downsteam region of thrBC genes were
amplified from MG1655 genome using the primer pairs thrAdn_OL /thrAF_isceI_sac
and thrCdn_IsceI_psti/thrCF_OL. The fragments were joined by over-lap PCR with
an EcoRV site separating them added by the primers. Then the joined fragment was
cloned into pMD19-T simple. The plasmid obtained was named pMD19-thrBCdel.
A 1.1 kb PCR fragment from pMDICI using primers mdF/mdR containing
chloroamphenicol resistance gene flanked by two I-SceI recognition sites was blunt
cloned into pMD19-thrBCdel at EcorV site, to yield pMD19-thrBC-Cm.
A 1.7 kb PCR fragment was amplified from pMD19-thrBC-Cm, using the primers
pair M13F / M13R. This fragment was transformed into the recipient strain by
electroporation
Donor plasmid with three fragments.
Donor fragments for cadA deletion and gdhA integration were amplified using the
primer pair cadAF_ol/cadAR_sacspe and gnttF_kpnXho/gntTR_OL and joined by
over-lap PCR. Then the joined PCR fragment was subcloned into pKSI-1 at PstI/XhoI
sites, yielding pKSIcgg.
0.7kb SacI/PstI fragment from pMD19-thrBCdel for thrBC deletion was then
subcloned into pKSIcgg, yielding pKSIcggt. The final resulting plasmid pKSIcggt
were used as the donor plasmid used for marker recycling and genetic modification.
(Fugure 2A)
Marker recycling and modification
To regenerate the marker, donor plasmid was transformed into an intermediate
strain with helper plasmid pREDTKI.*
Several resulting colonies were first incubated in LB liquid medium with 0.5%
glucose. Forty microliters of overnight seed culture were inoculated in a test tube
containing 4 mL LB medium with 50μg/mL kanamycin and 10mmol/L IPTG and the
test tube was cultivated at 30 ºC and 200 rpm for ~2 h. IPTG was added to the mixture
to a final concentration of 20 mmol/L and the test tube was cultivated for 6~8 h or
overnight. Forty microliters of culture were inoculated into another test tube
containing 4 mL LB medium with 20 mM IPTG, 10mM L-arabinose and 50μg/mL
kanamycin. The test tube was cultivated for 6~8 h or overnight. The culture was
harvested, suspended in sterile water, diluted and spread on LB plates supplemented
with 20mmol/L IPTG, 10mmol/L L-arabinose and 50μg/mL kanamycin. Colonies
appeared after overnight incubation at 30 ºC. Then the colonies were analyzed for
sensitive phenotype, and the final clones were confirmed by PCR.