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Strain Maintenance
STANDARD STRAINS
K802
Provenance: Wood, J. Mol. Biol. 16, 118–133 (1966).
Chromosome: galK2 galT22 metB1 lacY1(?) supE44 hsdr2
Sex: F–
Phenotype: Gln-inserting amber suppressor; restrictionless but modification plus; requires
(only) methionine and thiamine in minimal; good at propagating fd-tet family; the Lac
genotype is reported as “lac-3 or lacY1”; I’m designating it as lacY1 because it
behaves as a LacY (permease) defective mutant, in that it can’t ferment lactose, it is
white on Xgal plates, and it is blue on IPTG/Xgal plates.
NOTE: We no longer distribute K802, instead using MC1061 as the generic F– strain.
K91
Provenance: Derived in N.D. Zinder’s lab by curing K38 (Lyons and Zinder, Virology 49,
45–60, 1972) of  prophage
Chromosome: thi
Sex: Hfr-C (Cavalli)
Phenotype: stable Hfr, even without selection; excellent for propagating filamentous
phage; gives large plaques with wt phage, small but visible plaques with infective
members of fd-tet family; requires (only) thiamine in minimal medium
NOTE: We don’t routinely distribute this strain.
K91Kan
Provenance: our lab (p. SE94)
Chromosome: Same as K91, except that the mini-kan hopper element of Way et al. (Gene
32, 369–379, 1984) is inserted in the lacZ gene using the vector NK1105; the 9-bp
target site sequence CGCGTGGCA is centered on position 2527 relative to the start of
the lacZ start codon at 1). Once it has hopped, this transposon is stable since it doesn’t
carry its own transposase.
Sex: same as K91 (Hfr-C)
Phenotype: same as K91 except: LacZ-; kanamycin resistant
NOTE: We don’t routinely distribute this strain.
K91BluKan (B91BK)
Provenance: Cindy Schopp, our lab (p. CS1.78)
Chromosome: same as K91Kan except: mini-kan hopper is in the lacY gene rather than
lacZ; the 9-bp target site sequence TACATCACA is centered on position 105 relative
to the start of the lacY start codon at 1); lacZM15; probably is lacIQ
Sex: same as K91 (Hfr-C)
Phenotype: same as K91Kan, except: LacY-; probably LacIQ (super-repressor) lacZ donor
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K91Y(DE3)
Provenance: A K91 derivative constructed in our lab (K91deltaYDE34.doc)
Chromosome: thi lacY::IS5 DE3; same as K91 except:
 Lysogenic for the specialized transducing phage DE3 (Novagen), which contains
an IPTG-controlled T7 RNA polymerase gene
 An IS5 insertion in the lacY gene; the CTAG target site duplication for the
insertion starts with the CTA codon for the Leu at amino acid position 114
(relative to the initiating f-Met as position 1); the direction of the insertion is
“forward,” with the 338-codon transposase coding sequence in oriented in parallel
with the lacY coding sequence
Sex: same as K91 (Hfr-C)
Phenotype: When cultured in IPTG to induce expression of the T7 RNA polymerase
gene, this strain can express T7 promoter-driven genes (as in the pET expression
system). The lacY mutation is intended to uncouple induction from expression,
thus increasing the homogeneity of expression level across the entire bacterial
population at low concentrations of IPTG. Although IPTG can diffuse into the
cell without the aid of the lacY permease, it is nevertheless a substrate for that
permease (Hansen LH, Knudsen S, Sorensen SJ., The effect of the lacY gene on
the induction of IPTG inducible promoters, studied in Escherichia coli and
Pseudomonas fluorescens, Curr. Microbiol. 36, 341–347, 1998). Presence of an
active permease gene thus potentially results in a positive feedback loop in which
induction is coupled to expression. Such coupling can result in a heterogeneous
cell population at low inducer concentrations, some cells being fully induced
while the other cells are completely uninduced (Novick, A., and M. Weiner,
Enzyme induction as an all-or-none phenomenon. Proc. Natl. Acad. Sci. USA. 43,
553–567, 1957). In a permease-negative mutant like K91Y(DE3), in contrast,
entry of the IPTG inducer into the cell should be completely independent of
expression level.
NOTE: We don’t routinely distribute this strain.
MC1061
Provenance: Bill Dower, Affymax Research Institute; Meissner et al., Proc. Natl. Acad.
Sci. U.S.A. 84, 4171–4175 (1987).
Chromosome: hsdR mcrB araD139 (araABC-leu)7679 (lac)174 galU galK strA thi
Sex: FPhenotype: good host for electroporation; defective for EcoK restriction but
modification-proficient; defective for McrA and McrBC restriction systems; Lac–
(operon deleted, but I’m not sure whether lacI gene is functional); can’t
metabolize arabinose; can’t metabolize galactose; streptomycin resistant; requires
thiamine
TABLE OF PHENOTYPES
The Steps columns indicates the step in the protocol below where the indicated phenotype is
scored. With regard to the behaviors in this table, K91Y(DE3) resembles K91; the two strains
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can be easily differentiated in that the former is Lac– (unable to ferment lactose or to grow on
medium with lactose as the sole carbon and energy source) while K91 is Lac+.
Step
14
14
14
14
4
4
4
4
Test
Growth in following media
GB
GB + Kan
GB + Methionine
GB + Leucine + Strep
NZY
NZY + Kan
NZY + Strep
NZY + Tet
15
15
Plaque assays
M13mp2 + Xgal
plaques
lawn
M13mp2 + Xgal + IPTG
plaques
lawn
Diluent + Xgal + IPTG
plaques
lawn
16
16
Spot-test supernatants on
K91Kan-seeded plate
K91BK-seeded plate
15
15
15
15
K802
MC1061
K91Kan
K91BK
K91
Control
0
0
+
0
+
0
0
0
0
0
0
+
+
0
+
0
+
+
+
0
+
+
0
0
+
+
+
0
+
+
0
0
+
0
+
0
+
0
0
0
0
0
0
0
0
0
0
0
0
white
0
white
white
white
white
white
blue
white
0
blue
0
white
white
white
blue
white
blue
blue
0
blue
0
white
0
white
0
white
0
blue
NO
PLAQUES
plaque
plaque
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DAY 1
1. Make up 6 ml each of the following media with & without antibiotics and amino acids as
follows in 15-ml tubes or other sterile vessels; dispense 1 ml into each well of a row of a 24-well
culture dish as follows:
Dish 1, Row A:
Dish 1, Row B:
Dish 1, Row C:
Dish 1, Row D:
GB
GB+100 µg/ml Kanamycine
GB+100 µg/ml Streptomycin+20 µg/ml L-Leucine*
GB+20 µg/ml L-Methionine*
Dish 2, Row A:
Dish 2, Row B:
Dish 2, Row C:
Dish 2, Row D:
NZY
NZY+100 µg/ml Kanamycin
NZY+100 µg/ml Streptomycin
NZY+20 µg/ml Tetracycline
*L-Leucine and L-Methionine are prepared as 4 mg/ml stock solutions, autoclaved, and
stored at room temperature
2. Inoculate strains into all 4 wells of the indicated column of both dishes (8 wells altogether for
each column) as follows:
Column 1: K802
Column 2: MC1061
Column 3: K91Kan
Column 4: K91BlueKan
Column 5: K91
Column 6: leave uninoculated as control
3. Secure the dishes (Dish 1 and 2) in two separate humidified plastic boxes, shake gently at 37°
for 2 days (Dish 1) or 1 day (Dish 2).
NOTE: For Day 2, you’ll need at least 17 LB plates and TB soft agar.
DAY 2
4. Record growth pattern in the wells in Dish 2 , step 3 (with NZY medium); the pattern should
be that shown in the table above.
5. Use 10 µl of all 5 NZY cultures (one for each strain; without antibiotics) to inoculate 1 ml of
NZY in 15-ml tubes; shake vigorously at 37°.
6. Transfer the remainders of the 5 NZY cultures to sterile 1.5-ml Ep tubes; be sure to use
uncontaminated Ep tubes; keep these tubes on hand for use at step 12 below.
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7. Set up 20 tubes with 3 ml melted TB soft agar in a 50º temp-blok; within ~30 min of plating
(step 10 below) add 60 µl of 2% X-gal to 15 of the tubes and 25 µl of 0.2 M IPTG to 10 of the
tubes with X-gal.
8. Into 10 15-ml tubes on a slant pipette 10 µl of a dilution of M13mp2 phage (diluent =
TBS/gelatin) with an infectious unit concentration of ~120 pfu/10 µl. Into 5 more tubes on a
slant pipette 10 µl TBS/gelatin diluent as a control.
NOTE: Any “blue” phage (i.e., containing a lacZ  gene fragment for blue-white screening)
without amber mutations can be substituted for M13mp2.
9. When the 5 NZY cultures step 5 (one for each strain) have all grown, pipette 100 µl of each
culture into 3 of the slanted tubes at step 8: two tubes with M13mp2 and one control tube.
10. Plate the 3 slanted tubes for each strain in soft agar as follows:
One with M13mp2 in soft agar + Xgal + IPTG
One with M13mp2 in soft agar + Xgal (no IPTG)
One control tube (TBS/gelatin) in soft agar + Xgal + IPTG
Pour the soft agar into the 15-ml tube, and immediately pour onto an LB plate, tipping the plate
immediately to spread the soft agar.
11. Into two 15-ml tubes pipette 100 µl of the K91Kan and K91BlueKan cultures from step 5.
Pour each of these onto an LB plate in TB soft agar (without Xgal or IPTG) as in step 10.
12. Microfuge the 5 1.5-ml Ep tubes step 6 above (one from each strain) briefly. Pipette a 10-µl
droplet of each supernatant onto a spot of each of the two seeded plates step 11. As a positive
control, also pipette a 10-µl droplet of the mock library dilution (same as used in step 8) onto
each of the two seeded plates; use a separate pipette tip for each spotting to avoid any possibility
of contamination.
13. Incubate all 17 plates (15 from step 10, 2 from step 12) overnight at 37°.
DAY 3
14. Record growth pattern in the wells of Dish 1 step 3; they should match the pattern in the
table above.
15. Record the plaque pattern (including color) and lawn color in the 15 plates from step 10; the
pattern should match that in the table above.
16. Record whether there is a noticeable plaque at any of the spots on either of the two seeded
plates from step 12. There should be a marked plaque at the two positive control spots, and none
at the other spots, as noted in the table above.
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GB Medium
1. In a glass 125 ml bottle mix:
6.61 mmol KH2PO4 (e.g., 0.90 g anhydrous)
11.68 mmol Na2HPO4 (e.g., 1.66 g anhydrous or 3.13 g heptahydrate)
3.74 mmol NH4Cl (0.2 g)
99 ml water
2. Dispense 20 ml into each of 5 30-ml Nalge bottles.
3. Autoclave; when cool, add 176 µl of sterile GB Supplements (below) to each vial; check the
GB Supplements carefully for contamination before using it. Close caps tightly and store at
room temperature.
GB Supplements
1. Into 32.3 ml water in a beaker stirring vigorously on a magnetic stirrer, add 17.7 g glucose
(this makes 40% w/v glucose). Autoclave in a glass 125-ml bottle.
2. At the same time autoclave:
25 ml 0.4 M MgSO4 in a plastic bottle
3 ml 0.1 M CaCl2 in a polypropylene tube or bottle
10 ml 1% thiamine.HCl (vitamin B1) in a polypropylene tube or bottle
3. When all components are cool, add the three solutions from step 2 to the glass bottle from
step 1. This is GB Supplements. Store at room temperature.