Gene, 163 (1995) 35-40
© 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50
35
GENE 09127
Short Communications
Construction of laclts and laclqts expression plasmids and evaluation of
the thermosensitive lac repressor
(Recombinant DNA; cloning; lacI; lacZ; lacO; lacZop)
Noaman Hasan and Waclaw Szybalski
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, W1 53706-1599, USA
Received by J. Messing: 4 January 1995; Revised/Accepted: 15 May 1995/18 May 1995; Received at publishers: 2 June 1995
SUMMARY
To characterize a thermosensitive laclts mutant isolated by Bukrinsky et al. [Gene 70 (1989) 415-417] and to adapt
it as a convenient control element, we have (i) mapped the mutation to the inducer-binding domain of the LacIts
repressor, (ii) determined that the mutation resulted in the loss of a HaeIII site in laclts due to a G ~ A transition,
leading to a Gly ls7 ~Ser substitution, (iii) removed extraneous lacZop DNA downstream of lacI, and (iv) cloned laelts
(in plasmids based on ori of either ColE1 or P15A) under control of the wild-type or lacI q promoters. The LacIts
repressor is insensitive to IPTG. The repression of lacZop by LacIts is very efficient at 30°C and total induction was
achieved at 42°C, providing that the LacIts concentrations are not excessive and that repressor-to-operator ratios are
not too high.
INTRODUCTION
Many E. coli expression vectors use the lacZop operator/promoter present on multicopy plasmids. To insure
tight regulation of cloned genes, such plasmids must be
propagated in special host strains (lacI q) that overproduce LacI. This is of great importance when some of
the cloned gene products are detrimental to the host cell.
Inadequate levels of the LacI may lead to undesirable
Correspondence to: Dr. W. Szybalski, McArdle Laboratory, 1400
University Ave., Madison, WI 53706, USA. Tel. (1-608) 262-1259;
Fax (1-608) 262-2824; e-mail: [email protected]
Abbreviations: A, absorbance (1 cm); aa, amino acid(s); Ap, ampicillin;
[3Gal, [3-galactosidase; Cm, chloramphenicol; ENase, restriction endonuclease; GalK, galactokinase; galK, gene encoding GalK; hcn, high
copy number; IPTG, isopropyl-13-D-thiogalactopyranoside; kb, kilobase(s) or 1000 bp; lacl, gene encoding lac repressor; lacZop operator/
promoter of the lac operon; LB, Luria-Bertani (medium); mcn, medium
copy number; nt, nucleotide(s); ONPG, o-nitrophenyl-13-D-galactopyranoside; ori, origin(s) of DNA replication; p, plasmid; p, promoter;
5, resistant/resistance; Tc, tetracycline; ts, thermosensitive; wt, wild type;
XGal, 5-bromo-4-chloro-3-indolyl-13-D-galactopyranoside; [], denotes
plasmid-carrier state.
SSDI 0378-1119(95)00409-2
expression of cloned genes and to plasmid instability,
including selection against the desired clone.
LacI is a tetrameric molecule with three binding
domains, one (aa 1-59) recognizing the lacZo operator
DNA, the second recognizing the inducer, and the third
(aa 280-360) responsible for tetramerization (Miller and
Schmeissner, 1979; Miller et al., 1979).
The aim of the present study was to characterize the
laclts gene and its thermosensitive LacI product
described by Bukrinsky et al. (1989), and to construct
two sets of plasmids that overexpress the laclts gene, one
set of plasmids derived from pACYC184 that is compatible with ColEl-derived expression vectors containing
lacZop and the other set derived from plasmid pKO3.
When using the LacIts repressor, the gene expression
from the lacZop vectors will be inducible by heat rather
than by IPTG.
EXPERIMENTAL AND DISCUSSION
(a) Mapping and sequencing the laclts mutation
During preliminary analysis of the lacIts fragment we
noticed that it is missing the HaelII site(GGCC) over-
36
pNH321aclq
E
pNH371aclqts AE
laclqp
~1 ~I
E
lacl
lacits ~ ~
E
..~
laclts + ~
E
E
_.~
iaclqp
lapping aa residues Gly 187 and Pro 188 (Coulondre and
Miller, 1977). Sequence analysis the BstEII-HinclI fragment and the substitution of both terminal fragments
(EcoRI-BstEII and HinclI-EcoRI fragments) with the
analogous wt lacI DNA have indicated that the sequence
5'-CGGGCCC present in wt lacI + was changed to
5'-CGAGCCC in laclts. The G ~ A transition resulted in
the loss of the HaelII site, and in the Gly187~Ser
substitution.
Bs Ilc
.~
laclqp
pNH36/aclqts
E
pNH35/ac/ts
E
~.
[aclts v~ v~
pNH34 laclts
E
~
laclts ~ ~ll a ~ lacZ'
A
(b) Construction of plasmids containing either laclts or
laclqts
We have constructed several plasmids carrying laclts
in pKO3 (Fig. 1 A) and pACYC184 (Fig. 1B). The laclts
is either under the control of the wt lacI promoter or the
stronger lacI q promoter (Calos, 1978). For higher levels
of expression, the mutant lacI in plasmids pNH391aclts
and pNH411aclqts is also transcribed by the Cm R
promoter in addition to wt lacI or lacI q promoters,
respectively. Furthermore, the laclts in pNH381aclts
and pNH4Olaclqts is transcribed in the anti-sense
orientation by CmR-controlling promoter. Plasmids
pNH381aclts,
pNH391aclts,
pNH4OlacIqts
and
pNH411aclqts derived from pACYC184 (Fig. 1B) are
compatible with ColEl-derived plasmids.
~'0'
ApR
pKO3
galK
4.00 kb
ori
N
pNH39 laclts
pNH41 laclqts
E
laclpllaclqp " ~
,,4.- iaclpllaciqp
E
pNH38 laclts
B
p
Fig. 1. Construction of laclts and laclqts plasmids. Plasmids derived
from pKO3 (A: hen) or pACYC184 (B: mcn) and containing either
laclts or laclqts were constructed in a series of steps starting with pMCts
plasmid (M.I. Bukrinsky, personal communication) containing 1.7-kb
EcoRI fragment cloned in the EcoRI site of plasmid pBR322 (Bolivar
et al., 1977), encoding the laclts under the control of the wt lacl promoter, and carrying the lacZop downstream. (A) The 1.7-kb EcoRI
fragment from pMCts was cloned in the EcoRI site of plasmid pKO3
(McKenney et al., 1981) resulting in plasmid pNH341aclts. To remove
the lacZop, the 789-bp HinclI-EcoRI fragment ofpMCts was replaced
with the analogous 291-bp DNA fragment obtained from plasmid
pACYClacI q (J. Wang, personal communication), and the resulting
1.2-kb laclts fragment was cloned in the EcoRI site of plasmid pKO3,
to form pNH351aclts. To replace laclp with the strong laclqp promoter
(Calos, 1978), the EcoRI-BstEII fragment of pNH351aclts was replaced
by the analogous fragment of pACYClacl q, to form pNH361aclqts. The
EcoRI site present upstream of the laclts in pNH361aclqts was filledin resulting in plasmid pNH371aclqts that contains only one downstream EcoRI site. Plasmid pNH321acl q was constructed by cloning the
1.2-kb EcoRI fragment from pACYC1841acl q in the EcoRI site of plasmid pKO3. The two small downward arrows indicate the approximate
location of the BstEII and HinclI sites within lacI. B, BglI; Bs, BstEII;
E, EcoRI; H, HindlII; N, NdeI; P, PstI; S, Sinai; X, XmnI. X 'O' is a
remnant of the L O gene truncated at both ends. (B) The 1.2-kb EcoRI
fragments of pNH351aclts and pNH361aclqts were cloned in the EcoRI
site of mcn plasmid pACYC184 (Chang and Cohen, 1978). Plasmids
pNH381aclts, pNH391acIts, pNH4Olaclqts and pNH411aclqts that are
compatible with ColEl-derived expression vectors were obtained. The
lacI gene in pNH381aclts and pNH4Olaclqts is in the orientation opposite to the Cm a gene, whereas that in pNH391aclts and pNH411aclqts
is in the same orientation as that of Cm R gene. The lacIts is transcribed
in the anti-sense orientation by Cm R promoter (marked with p and a
small arrow) in plasmids pNH381aclts and pNH4Olaclqts, but in the
sense orientation in plasmids pNH391aclts and pNH411aclqts, both by
the Cm R and its own promoters. The orientation of all the genes and
constructs was determined by restriction analysis.
37
(c) Heat inactivation of LacIts
repression/derepression in cells containing mcn plasmids
carrying laelts.
To test the importance of lacZop/LacIts ratio in the
thermal induction of lacZop, we measured chromosomal
expression in the presence of pNH258 (Hasan and
Szybalski, 1986), a multicopy plasmid derived from
pKO3 and carrying lacZop. We found that full thermal
induction of chromosomal lacZ gene was achieved even
in the presence of multicopies of lacZop (Table II).
Similarly, complete induction of lacZop-promoted galK
expression was obtained in strain C600[pNH455-1]
transformed with the same laclts plasmids (pNH381aclts,
pNH391aclts, pNH4Olaclqts and pNH411acIqts). Plasmid
pNH455-1 is a multicopy plasmid that contains lacZopnutL-N-tLl-galK. As shown in Table III, complete induction of galK gene expression was obtained at 42°C.
Furthermore, plasmids pNH391aclts, pNH4Olaclqts and
pNH411aclqts produce sufficient laclts repressor to completely repress multicopies ( ~ 5 0 - 1 0 0 copies) of lacZop
at 30°C. In contrast, plasmid pNH381aclts expresses the
repressor at lower levels that results in only partial repression of multicopies of lacZop. Collectively, these results
indicate that repression of lacZop by laclts was complete
at 30°C and that total induction was achieved at 42°C,
providing the repressor-to-operator ratios were not
excessively high (Tables II and III).
On XGal plates, similar results were obtained
with pMCts and only pNH341aclts (as with pNH38lacIts
and pNH401acIqts). Both LBG1081 [pNH341aclts] and
LBG1081 [ p M C t s ] gave white colonies at 30°C and blue
colonies at 42°C. In contrast, LBG1081[pNH351acIts]
and LBG1081[pNH361acIqts] produced white colonies
at 30°C and 42°C (not shown). The results were
We tested heat inactivation of LacIts by assaying chromosomal lacZ expression. Heat inactivation of LacIts
was
studied
using
pNH381acIts, pNH39lacIts,
pNH4OlacIqts and pNH411acIqts. The plasmids were
tested in strain LBG1081 that is deficient in LacI
(Bolshakova et al., 1978). Cells transformed with these
plasmids were initially plated on LB agar + XGal plates
suplemented with the 10 gg Tc/ml at both 30°C and 42°C.
We found that LBG1081 transformed with either
pNH381acIts or pNH4OlacIqts gave white colonies on
XGal plates at 30°C and blue colonies at 42°C, whereas
cells
transformed
with
either
pNH391acIts or
pNH411acIqts gave white colonies on XGal plates at both
30°C and 42°C. The lacIts in pNH38lacIts and
pNH40IacIqts is transcribed only by its own promoter
(and by the Cm a promoter in the anti-sense
orientation), whereas the lacIts gene in pNH39lacIts and
pNH411acIqts is transcribed in the sense direction by
both the Cm a promoter and its own promoter (Fig. 1B).
Unlike for cells transformed with pACYC1841acIq, the
addition of I P T G to XGal plates had no effect on the
colony phenotype at both temperatures (Table I).
Assays of the chromosomal [3Gal (Table II) for
LBG1081 [pNH381aclts] and LBG1081 [pNH4Olaclqts]
were positive at 42°C but not at 30°C, whereas LBG1081[pNH391aclts] and LBGlOSl[pNH411aclqts] did not
express significant levels of [3Gal at either temperature.
Unlike for cells containing wt LacI, the level of [3Gal
expression was not influenced by the presence or absence
of IPTG. The lack of expression of 13Gal in the presence
of pNH391acIts and pNH411aclqts suggested that the
gene dosage of laclts plays an important role in lacZop
TABLE I
Effect of heat, IPTG and lactose on the repression by LacIts. Phenotypes of E. colistrains D1210 and LBG1081 in the presence of laclts plasmids
grown on various indicator platesa
Plasmidsb
None
pNH381aclts
pNH391aclts
pNH4Olaclqts
pNH41laclqts
IPTG +XGaF 30/42°C
MacConkey+lactosed 30°C
Tetrazolium + lactosee 30°C
2
3
4
5
6
7
D1210
LBG1081
D1210
LBG1081
D1210
LBG1081
blue/blue
blue/blue
blue/blue
white/blue
white/blue
blue/blue
white/blue
white/blue
white/white
white/white
red
white
white
white
white
red
white
white
white
white
white
red
red
red
red
white
red
red
red
red
a The cells weregrown overnightat 30° or 42°C on indicator plates containingthe appropriate concentration of antibiotics as described by Miller (1972).
b Plasmids are described in Fig. 1.
c The colonyphenotype is indicated under each strain first at 30°C and then at 42°C. E.g., as indicated in column 3 for strain LBGI081[pNH381aclts],
the white/blue phenotypes indicate that the colonies are white at 30°C and blue at 42°C.
d Red and white indicate the phenotype of the resulting colonies in the presence or absence of lacZinduction, respectively.
e Red and white indicate the phenotype of the resulting colonies in absence or presence of lacZinduction, respectively.
38
TABLE II
Expression of chromosomal l a c Z as controlled by the presence of lacIts plasmids and multicopies of lacZop
Plasmida
Presence of
pNH258 b
[3Gal induction (%)c
+ I P T G (30°C)
pNH381ac lts
-
+
pNH391acIts
-
+
pNH4Olaclqts
-
+
- I P T G (30°C)
(control)
0
0
ND
25
0
0
ND
2
0
0
ND
1
0
0
- I P T G (42°C)
100
100
20
100
100
100
19
100
100 (880)
100
pNH411aclqts
-
pACYC184
+
+
ND
100
ND
pACYC 1841acIq
+
1O0
0
0
pMCts
-
0
0
pNH341aclts
-
0
0
100
100
pNH351aclts
-
0
0
0
pNH361aclqts
-
0
0
0
pNH371aclqts
-
0
0
pKO3
-
100
2
100 (710)
100
100 (835)
0
100 (994)
a Plasmids are described in Fig. 1.
b Presence (+) or absence ( - ) of multicopy, pKO3-derived pNH258 containing lacZop (Hasan and Szybalski, 1986).
° E. coil strain LBG1081 that is l a c I - lacZop + l a c Z + (Bolshakova et al., 1978) was used for ~Gal assay. Cells containing the specified plasmids were
incubated at either 30° or 42°C for 3 h in LB containing 10 Ixg Tc/ml, in the presence or absence of 1 mM IPTG. The toluene-treated cells were
prepared as described (Hasan and Durr, 1974) and 13Gal levels were determined by ONPG hydrolysis (Lederberg, 1953) and expressed as percentage
of the levels obtained in cells harboring control plasmid pACYC184 E710 and 880 nmol (see in parentheses) of ONPG hydrolyzed per min per A65onm
using cells grown at 30°C and 42°C, respectively, in the absence of IPTG]. The values obtained with plasmids pMCts, p N H 3 4 1 a c l t s , p N H 3 5 1 a c l t s ,
p N H 3 6 1 a c l q t s and p N H 3 7 1 a c l q t s were expressed as a percentage of that obtained with plasmid pKO3 E835 and 994 nmol (see in parentheses) of
ONPG hydrolyzed per min per A65o nm using cells grown at 30°C and 42°C, respectively, in the absence of IPTG]. ND, not determined.
confirmed by assaying 13Gal expression in the presence
or absence of I P T G (Table lI). In the presence of
pNH351aclts, pNH361aclqts or pNH371aclqts, complete
repression of chromosomal [3Gal was observed at both
temperatures in the presence or absence of IPTG. The
laclts in these plasmids is missing the lacZop sequence
naturally present downstream of lacI in plasmids pMCts
and pNH341aclts. The lack of 13Gal expression in the
pres.ence of the former three plasmids further indicated
the importance of laclts gene dosage in lacZop repression/derepression.
(d) IPTG and lactose induction of IIGal
The laclts mutation is in the core of the lac repressor
protein that contains the inducer-binding domain (Miller,
1980). As shown in Table II, LBG1081 cells containing
the lacIts or laclqts did not express appreciable levels of
[3Gal in the presence of I P T G at 30°C. However, at 42°C,
13Gal expression was observed in the presence or absence
of IPTG. The lack of induction with I P T G was observed also for pMCts, pNH341aclts, pNH351aclts and
pNH361aclqts (Table II). Unlike wt LacI, the LacIts is
refractory to induction by I P T G possibly due to a
defective repressor-inducer interaction. 13Gal was fully
induced by I P T G in LBG1081 cells transformed with
plasmids carrying wt lacl q (Table II). The ts phenotype
was recessive following the introduction of plasmids carrying laclts and laclqts into E. coli strains carrying wt
lacIq. Furthermore (Table I), following the introduction
of the laclts-containing
plasmids (pNH381aclts,
pNH391aclts, pNH4Olaclqts and pNH411aclqts) into E.
coli strain D1210 (Sadler et. al., 1980) that contains a
chromosomal copy of wt lacIq, the chromosomal lacZ
was induced by I P T G at both 30 ° and 42°C in strains
containing pNH381aclts and pNH391aclts and only
at 42°C in strains containing pNH4Olaclats and
pNH411aclqts. This was evident on L B + X G a l + I P T G
plates, as blue colonies were obtained at 42°C in strains
harboring any of the four laclts-containing plasmids. On
the other hand, only strains containing pNH381aclts and
pNH391aclts but not pNH4Olaclqts or pNH411aclqts
gave blue colonies at 30°C. Moreover, lactose was not
an inducer of chromosomal lacZ in either strain D1210
or LBG1081 containing the laclts plasmids, since in both
strains and in the presence of any laclts plasmid, white
colonies were obtained at 30°C on lactose/MacConkey
39
TABLE III
Heat induction of lacZop-promoted galK expression as controlled by
the presence of either laclts or laclqts
Plasmida
pNH381aclts
pNH391aclts
pNH4Olaclqts
pNH411aclqts
GalK (units) b
30°C
42°C
24
8
2
0.5
284
275
328
318
The plasmids and orientations of laclts inserts are the same as
described in Table II and Fig. 1.
b E. coli C600(galK-) was transformed with the plasmids indicated in
the first column and selected on LB containing 10 ~g Tc/ml. The cells
were subseqently transformed with plasmid pNH455-1 (Hasan and
Szybalski, 1986) containing lacZop-nutL-N-tLl-gaIK module, and the
cells were grown overnight on LB containing 10 pg Tc/ml and 50 pg
Ap/ml at 30°C, diluted 1:100 into fresh L B + A p + T c medium, and
grown either at 30°C or 42°C for 3 h (A650nm 0.4--0.6). Samples of 1 ml
were treated with toluene, and GalK activity assayed as reported in
Hasan and Szybalski (1986). GalK units are expressed as nmol of
[14C]galactose phosphorylated per min per A65onm. Control sample
used was E. coli strain C600 carrying plasmids pKO3 and pNH381aclts,
and the GalK values of 2 and 4 units obtained at 30°C and at 42°C,
respectively, were subtracted from the values obtained with experimental samples.
plates (Table I). Furthermore, plating D1210 and
LBG1081 cells containing any of the laclts plasmids on
lactose/tetrazolium agar gave only red colonies at 30°C,
further indicating the lack of induction of 13Gal by lactose
(Table I). Strain LBG1081 lacking the laclts plasmids
and strain D1210 containing chromosomal lacIq, gave
red and white colonies on lactose/MacConkey and lactose/tetrazolium plates, respectively, at 30°C indicating
that both strains contained a functional lacZ gene, and
that 13Gal was induced by lactose in D1210. IPTG
induces lacZ at 30°C in strain D1210 containing
pNH381aclts and pNH391aclts but not pNH4Olaclqts
and pNH411aclqts plasmids. Furthermore, IPTG did not
induce chromosomal lacZ in strain LBG1081 carrying
the laclts or laclqts plasmids in absence of wt lacI gene.
The above results are summarized in Table I. Collectively,
the data suggest that Laclts repressor monomers form
hybrid tetramers with wt LacI repressor monomers and
this hybrid repressor can be inactivated by IPTG but
not lactose.
(e) Conclusions
(1) We have constructed two sets of plasmids derived
from hcn pKO3 and mcn pACYC184 carrying the laclts
gene. In addition to the laclp or laclqp, the laclts in
pNH391aclts and pNH411aclqts is also transcribed by
the Cm R promoter. The level of LacIts produced in the
presence of some of the plasmids is capable of completely
repressing lacZop present on hcn compatible plasmids.
Derepression of the lacZop is achieved only by heating
the cells at 42°C to inactivate the ts repressor. The mutant
repressor is insensitive to IPTG or lactose induction. The
host strain to be used with these plasmids is a strain
deficient in LacI such as strain LBG1081. However, in
wt E. coli strains, the level of repression of lacZopcontrolled gene expression by LacI could be tolerated
particularly in cases of hcn plasmids, as evident from the
results presented in Table III.
(2) The plasmids described in this report would eliminate the need for chemical inducers particularly in large
cultures. Furthermore, the use of the ts repressor constructs provides an advantage over the wt repressor in
that the mutant repressor will be insensitive to inducers
which might be present as contaminant (lactose-like) in
culture media, and that would provide tighter repression
of cloned genes.
(3) Plasmids pNH381aclts, pNH4Olaclqts and
pNH341aclts are the plasmids of choice for single
copy lacZop-controlled gene expression, whereas
pNH4Olaclqts and pNH411aclqts are the plasmids of
choice for multicopy vectors carrying lacZop-controlled
genes.
(4) The plasmids derived from pKO3 could be used to
construct expression vectors where both the lac repressor-encoding gene and lac promoter/operator are present
on the same plasmid.
ACKNOWLEDGEMENTS
Plasmid pMCts was a gift from Dr. M.I. Bukrinsky.
This work was supported by the NIH grants
1-RO1-GM39715 to W.S. and the NCI Core Grant
5-PO1-CA0.7175.
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