Table 1 Bacterial strains and plasmids used in this study
From: A genetic replacement system for selection-based engineering of essential proteins
Bacterial strain or plasmid | Characteristics | Source |
|---|---|---|
Strains | ||
W3110 | F- λ-rph-1 INV(rrnD, rrnE) | [33], internal strain collection |
DH10B | F-endA1 recA1 galE15 galK16 nupG rpsL ΔlacX74 Φ80lacZΔM15 araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) λ- | [34], internal strain collection |
W3110 adk::kan | Chromosomal adk replaced by a KmR cassette. The strain is only viable if the adk deletion is complemented. | This work |
W3110 groE::kan | Chromosomal groS and groL replaced by a KmR cassette. The strain is only viable if the gro E deletion is complemented. | This work |
W3110 secB-gpsA::kan | Chromosomal secB and gpsA replaced by a KmR cassette. The strain is only viable if the gps A deletion is complemented. | This work |
SBΔrecA | lacIqrrnB3 ΔlacZ4787 hsdR514 Δ (araBAD) 567 Δ (rhaBAD) 568 rph-1 groE::P araBAD -groE-KmRrecA::FRT | This work |
Plasmids | ||
pKD46 | ori pSC101ts, ApR. Encodes λ red recombination genes γ, β and exo under control of the arabinose-responsive promoter Para BAD. | [1] |
pKOCOMP-adk | pKOCOMP-derived vector with adk under control of an IPTG-responsive tac promoter | This work |
pSEVA432 | ori pBBR1, SpecR, multiple cloning site | provided by Victor de Lorenzo |
pCOMP-adk | pSEVA432 encoding for adk under control of an IPTG-responsive tac promoter, contains an I-Sce I cleavage site | This work |
pCOMP-ESL | pSEVA432 encoding for groS and groL control of an IPTG-responsive tac promoter, contains an I-Sce I cleavage site | This work |
pCOMP-sec B-gps A | pSEVA432 encoding for the natural secB-gpsA transcriptional unit, contains a I-Sce I cleavage site | This work |
pSEVA132 | ori pBBR1, ApR, multiple cloning site | provided by Victor de Lorenzo |
pSEVA132-adk | pSEVA132 encoding for adk under control of its natural promoter | This work |
pSEVA132-adkStop | pSEVA132-adk with an internal stop codon | This work |
pSEVA132-adkwatermark | pSEVA132-adk with a peptide insertion behind position P140 | This work |
pSEVA132-gro E | pSEVA132 coding for groS and groL under control of their natural promoter | This work |
pSEVA132-gro EStop | pSEVA132-groE with an internal stop codon | This work |
pSEVA132-gro Ewatermark | pSEVA132-groE with a peptide insertion behind site I301 | This work |
pSEVA132-sec Bgps A | pSEVA132 encoding for secB and gpsA under control of their natural promoter | This work |
pSEVA132- sec Bgps AStop | pSEVA132- sec Bgps A with an internal stop codon | This work |
pSEVA671 | ori p15A, GmR, multiple cloning site | provided by Victor de Lorenzo |
pI-Sce I | pSEVA671, with I-Sce I nuclease under control of the rhamnose inducible promoter PRha and the response regulators RhaS and RhaR, derived from the rhammnose metabolizing transcriptional unit of E. coli. | This work |
pParaI-Sce I | pI-Sce I with PrhaBAD and regulators RhaS and RhaR exchanged by the arabinose promoter Para BAD and the regulator araC | This work |