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. 2018 Oct 30;7:e34878. doi: 10.7554/eLife.34878

Key resources table. Genetic reagents, bacterial strains, antibodies, and software used in this study.

Reagent
type (species)
or resource		 Designation Source or
reference		 Identifiers Additional
information		 Isaacs
Lab
Reference
#		 Full
genotype
of strains		 # UAG
Codons		 RF1
status		 Ribosomal
rescue
gene
knockout		 ssrA
tag
status
Gene
(Escherichia
coli)		 pUAG-GFP this paper eGFP-6xHis
-UAG; Plasmid
NJM88;
Strain
NJM1242		 eGFP protein
with a C-terminal
6-His tag for protein
purification,
terminating
translation in a
UAG codon.		 Plasmid
NJM88;
Strain
NJM1242		 N/A N/A N/A N/A N/A
Gene
(E. coli)		 pUAA-GFP this paper eGFP-6xHis
-UAA; Plasmid
NJM89;
Strain
NJM1249		 eGFP protein with
a C-terminal 6-His
tag for protein
purification,
terminating
translation in a
UAA codon.		 Plasmid
NJM89;
Strain
NJM1249		 N/A N/A N/A N/A N/A
Genetic
reagent
(E. coli)		 RK24 10.1126/science
.1205822;
10.1016/j.cels
.2016.06.009		 pRK24;
Strain NJM699		 Conjugative RK2
plasmid (10.1006/
jmbi.1994.1404),
but lacks functional
AmpR gene.		 Strain
NJM699		 N/A N/A N/A N/A N/A
Genetic
reagent
(E. coli)		 F Yale University
Coli Genetic
Stock Center
(CGSC),
Strain #4401		 pF; Strain
EMG2; Strain
CGSC#4401;
Strain
NJM426;
Strain
NJM473		 Conjugative F
plasmid, as
described by
PMID: 4568763.
Obtained from
the Yale CGSC.		 Strain
NJM426;
Strain
NJM473		 N/A N/A N/A N/A N/A
Genetic
reagent
(E. coli)		 pZE21_
UAG-GFP		 this paper pZEtR-eGFP
-cHis-TAG-
v02; Plasmid
NJM88;
Strain
NJM1242		 pZE21 plasmid
with pLtetO
promoter driving
inducible expression
of eGFP with a
C-terminal 6-His
tag and terminating
in UAG codon.
Inducible with
anhydro-tetracycline.		 Plasmid
NJM88;
Strain
NJM1242		 N/A N/A N/A N/A N/A
Genetic
reagent
(E. coli)		 pZE21_
UAA-GFP		 this paper pZEtR-eGFP
-cHis-TAA-v02
; Plasmid
NJM89;
Strain
NJM1249		 pZE21 plasmid
with pLtetO
promoter driving
inducible expression
of eGFP with a
C-terminal 6-His
tag and terminating
in UAA codon.
Inducible with
anhydro-tetracy
cline.		 Plasmid
NJM89;
Strain
NJM1249		 N/A N/A N/A N/A N/A
Genetic
reagent
(Enteroba
cteria
phage λ)		 λ.CI857 Coli Genetic
Stock Center
(CGSC), Yale
University
(contact John
Wertz directly)		 λ.CI857; λ
phage;
Phage NJM102		 Phage λ with
temperature-
sensitive CI
repressor gene;
when incubated
at 37° C, phage
becomes obligate
lytic		 Phage
NJM102		 N/A N/A N/A N/A N/A
Cell line
(E.
coli)		 GRO.DD this paper C31GIB.
tmRNA-DD;
Strain #987		 MG1655-derived
strain with all 321
UAG codons
mutated to UAA,
deletion of RF1,
and tmRNA tag
C-terminal amino
acids mutated from
AA to DD. Retains
lambda red cassette
for recombineering.
Investigated in
Figure 2.		 Strain
#987		 ΔmutS:zeo.
Δ(ybhB-
bioAB)
:[λcI857.
Δ(cro-ea59)
:tetR-bla].
ΔprfA.ΔtolC
.tmRNADD 		0 +RF1 n/a DD
Cell line
(E. coli)		 GRO.
DD.prfA+		 this paper C31GIB.
prfA+.tmRNA
-DD; Strain
#996		 MG1655-derived
strain with all 321
UAG codons
mutated to UAA,
retains RF1,
and tmRNA tag
C-terminal amino
acids mutated from
AA to DD. Retains
lambda red cassette
for recombineering.
Investigated in
Figure 2.		 Strain
#996		 ΔmutS:zeo.
Δ(ybhB-
bioAB)
:[λcI857.
Δ(cro-ea59):
tetR-bla].
ΔtolC.tm
RNADD 		0 ∆RF1 n/a DD
Cell line
(E. coli)		 ECNR2 10.1016/j.cels
.2016.06.009		 ECNR2.Δmut
S:zeocin.Δ
λRed; Strain
#795		 MG1655-derived
strain that contains
321 UAG codons
and retains RF1.
Investigated in
Figures 3 and 4.		 Strain
#795		 ΔmutS:zeo 321 +RF1 n/a AA
Cell line
(E. coli)		 GRO.AA 10.1016/j.cels
.2016.06.009		 C31.final.
ΔmutS:
zeocin.ΔprfA
.ΔλRed;
Strain #796		 MG1655-derived
strain with all 321
UAG codons
mutated to UAA,
deletion of RF1.
Investigated in
Figures 3 and 4.		 Strain
#796		 ΔmutS:
zeo.ΔprfA
(GenBank
ID:
CP006698)		 0 ∆RF1 n/a AA
Cell line
(E. coli)		 GRO.
AA.∆arfB		 this paper C31GIB.arfB:
tolCorf.
ΔλRed;
Strain #1230		 MG1655-derived
strain with all 321
UAG codons
mutated to UAA,
deletion of RF1,
and deletion of arfB.
Investigated in
Figures 3 and 4.		 Strain
#1230		 ΔmutS:
zeo.ΔprfA
.arfB:tolC 		0 ∆RF1 ∆ssrA AA
Cell line
(E. coli)		 GRO.
AA.∆ssrA		 this paper C31GIB.ssrA
:tolC.ΔλRed;
Strain #1231		 MG1655-derived
strain with all 321
UAG codons
mutated to UAA,
deletion of RF1,
and deletion
of ssrA.
Investigated in
Figures 3 and 4.		 Strain
#1231		 ΔmutS:
zeo.ΔprfA.
ssrA:tolC 		0 ∆RF1 ∆arfA AA
Cell line
(E. coli)		 GRO.
AA.∆arfA		 this paper C31GIB.arfA
:tolC.ΔλRed
; Strain #1232		 MG1655-derived
strain with all
321 UAG codons
mutated to UAA,
deletion of RF1,
and deletion of
arfA. Investigated
in Figures 3 and 4.		 Strain
#1232		 ΔmutS:
zeo.ΔprfA.
arfA:tolC 		0 ∆RF1 ∆arfB AA
Cell line
(E. coli)		 GRO.AA
.∆ssrA.∆arfB		 this paper C31GIB.ΔarfB
.ssrA:tolC.Δ
λRed; Strain
#1233		 MG1655-derived
strain with all
321 UAG codons
mutated to UAA,
deletion of RF1,
and deletion of
ssrA and arfB.
Investigated in
Figures 3 and 4.		 Strain
#1233		 ΔmutS:
zeo.ΔprfA
.ΔarfB.ssrA:tolC 		0 ∆RF1 ∆ssrA.
∆arfB		 AA
Cell line
(E. coli)		 GRO.AA
.∆arfA.
∆arfB		 this paper C31GIB.Δarf
B.arfA:tolC.
ΔλRed;
Strain #1234		 MG1655-derived
strain with all
321 UAG codons
mutated to UAA,
deletion of RF1,
and deletion of
arfA and arfB.
Investigated in
Figures 3 and 4.		 Strain
#1234		 ΔmutS:
zeo.ΔprfA
.ΔarfB.arfA
:tolC 		0 ∆RF1 ∆arfA.
∆arfB		 AA
Antibody mouse
anti-GFP
antibody		 other Invitrogen
(Ref#: 332600,
Lot#:
1513862A)		 Invitrogen
(Ref#: 332600,
Lot#: 1513862A);
(5.5 μL antibody
in 3 mL Milk
 + TBST)		 N/A N/A N/A N/A N/A N/A
Antibody goat
anti-mouse
antibody		 other AbCam (Ref#:
ab7023, Lot#:
GR157827-1)		 AbCam (Ref#:
ab7023, Lot#:
GR157827-1);
(2.2 μL antibody
in 10 mL Milk
 + TBST)		 N/A N/A N/A N/A N/A N/A
Recombinant
DNA reagent		 ssrA:tolC this paper; for
use, see
tolC positive
/negative
selection in
10.1038/nprot
.2014.081		 dsDNA
NJM111		 The E. coli native
tolC gene used to
delete ssrA gene via
recombineering
(10.1038/nprot.
2008.227).		 dsDNA
NJM111		 N/A N/A N/A N/A N/A
Recombinant
DNA reagent		 arfA:tolC this paper; for
use, see
tolC positive
/negative
selection in
10.1038/nprot
.2014.081		 dsDNA
NJM112		 The E. coli native
tolC gene used to
delete arfA gene
via recombineering
(10.1038/nprot.
2008.227).		 dsDNA
NJM112		 N/A N/A N/A N/A N/A
Recombinant
DNA reagent		 arfB:tolC this paper; for
use, see
tolC positive
/negative
selection in
10.1038/nprot
.2014.081		 dsDNA
NJM113		 The E. coli native
tolC gene used to
delete arfB gene
via recombineering
(10.1038/nprot.
2008.227).		 dsDNA
NJM113		 N/A N/A N/A N/A N/A
Software,
algorithm		 Doubling
time
algorithm		 10.1126/
science.1241459		 Growth_
Analyze_
GK.m		 Doubling time
used in 10.1126
/science.1241459,
written by Gleb
Kuznetsov in the
lab of Dr. George
Church.		 N/A N/A N/A N/A N/A N/A
Software,
algorithm		 MaxQuant
v1.5.1.2		 other N/A Commercial
software for
mass
spectrometry
analysis.		 N/A N/A N/A N/A N/A N/A
Software,
algorithm		 Graphpad
Prism 7		 other N/A Commercial
software for
statistical
analysis and
graphing,
provided
through Yale
University.		 N/A N/A N/A N/A N/A N/A