[41]
COMPILATION OF SUPERLINKER VECTORS
469
[4 1] C o m p i l a t i o n o f S u p e r l i n k e r V e c t o r s
By J~3R~EN BROSIUS There are 64 possibilities for uninterrupted palindromic hexameric restriction enzyme recognition sequences. Enzymes for 52 of those sequences are commercially available. 1Many of the remaining 12 will follow over the next years. A large polylinker has been synthesized that accommodates all the above 64 restriction sites as well as 2 octameric (NotI and Sill) and a few interrupted palindromic and nonpalindromic hexamer sites.2 Several variants of the linker have been inserted into a number of popular multifunctional cloning and expression vectors. 2 These vectors will facilitate many cloning and subcloning applications because of their potential to accommodate restriction fragments irrespective of the enzymes by which they have been generated. In most cases these restriction sites will be preserved. Multifunctional Cloning Vectors Characteristics of some of the more frequently requested as well as four further developed cloning and expression vectors are summarized in Table I. pSL180, pSL190, pSLll80, and pSLI1902 correspond to commonly used vectors pUC18, pUC 19, pUC118, and pUC119. 3 The polylinkers of the latter group have been replaced with the SL1 version [317 base pairs (bp), flanked by EcoRI and HindIII] of the superlinker (Fig. 113). Because of the extended interruption of the LacZa' peptide derivative, a color discrimination between nonrecombinant and insert-containing vectors is not possible anymore. Therefore, bacterial colonies harboring those plasmids are white on indicator plates containing 5-bromo-4-chloro-3-indolyl-fl-D-galactopyranoside (X-Gal) and isopropyl-fl-o-thiogalactopyranoside (IPTG). pSL1180/pSL1190 differ from pSL180/pSL190 by the presence of the intergenic region (IR) from filamentous single-stranded phage M13, which contains the origin of replication as well as signals for phage packaging. This permits the generation of single-stranded DNA following superinfection with helper phage KO7 using a protocol described in an earlier volume of this series. 4 The single-stranded DNA corresponds, as I R. 2 j. 3 j. 4 j.
J. R o b e r t s a n d D. M a c e l i s , Nucleic Acids Res. 20(Suppl.), 2167 (1992). B r o s i u s , D N A 8, 759 (1989). M e s s i n g , Gene 100, 3 (1991). V i e i r a a n d J. M e s s i n g , this series, Vol. 153, p. 3.
METHODS IN ENZYMOLOGY, VOL. 2t6
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
470
VECTORS FOR CLONING GENES
[41]
TABLE I PLASMIDS W I T H SUPERLINKERS a
Size (bp)
Linker
Relevant features
2946
SL1
pSL190 b pUC19 c pSLI180 b'd pUC118 ~
2946 3422
SL1 SL1
pSL1190 b'd pUC119 e pSEI200 b pUC120f
3422 3442
SL1 SL2
pSL300 b
pBS KS(+)g
3264
SL2
pSL301 b,h
pSL300 b
3267
SL2
pSL350 i
pSE1200 b
3594
SL2
pSL351 i
pSL350i/ pUC181 j
3594
SL2
Cloning vector; no color screening for inserts As pSL180; linker in opposite orientation Cloning vector; no color screening for inserts; M 13 origin of replication for generation of single-stranded DNA (the coding strand with respect to the lacZ region) As pSL1180; linker in opposite orientation Cloning and expression vector; no color selection for inserts; M13 origin; lac promoter/operator; lac RBS; ATG start codon in NcoI site SL2 replaces linker from pBluescript KS M13(+); cloning vector; T3 and T7 transcription promoters flanking superlinker; fl origin of replication for generation of single-stranded DNA; translation restart distal to linker into lacZod; subtle color discrimination (midblue to light blue) for insert screening As pSL300; different translation restart from linker into lacZa'; subtle color discrimination (midblue to light blue) for insert screening Cloning and expression vector; T3 and T7 transcription promoters flanking superlinker; M13 origin of replication; lac promoter/operator; T7 gene 10 translational enhancer; minicistron upstream from ATG-containing NcoI site for translational restart; open reading frame throughout superlinker; distal to linker translational restart into wild-type lacZot; subtle color discrimination (blue to midblue) for insert screening As pSL350; M13 origin in opposite orientation; single-stranded DNA (yield significantly lower as with pSL350) representing the anticoding strand with respect to the lacZ region
Plasmid pSL180 b
Parent pUC18 c
[41]
COMPILATION OF SUPERLINKER VECTORS
471
TABLE I (continued)
Plasmid
Parent
Size (bp)
Linker
pKK489-7 ~ pSL350 i
3303
pSE220 b
pKK233-2 k
4926
SL2
pSE280 b,h
pSE220 b
3865
SL2
pSE380 b'h
pTrc99A /
4467
SL2
pSE420 i,h
pSE380 b
4617
SL2
pKK480-3 i
pSE420 i
4760
pKK520-3 i
pKK232-8 m
5417
SL2
Relevant features As pSL350; shorter polylinker (62 bp) from pTrc99AI; useful for future constructions requiring sites not present in the linker Expression vector; trc promoter; lac operator/RBS; inducible with IPTG, ATG in N c o I site; rrnB transcription terminators As pSE220; shorter, remnants of tet gene removed As pSE280; harbors in addition lac repressor (lacI) gene As pSE380; T7 gene 10 translational enhancer; A/T-rich regions flanking RBS; minicistron upstream from CCATGG ( = start codon in N c o I cloning site) for efficient translational restart; open reading frame throughout superlinker As pSE420; rat calmodulin gene instead of linker; model system for very high level of recombinant protein expression Promoter selection vector; linker upstream from promoter-deficient chloramphenicol acetyltransferase gene
All vectors confer resistance to ampicillin. b j. Brosius, D N A 8, 759 (1989). c j. Norrander, T. Kempe, and J. Messing, Gene 26, 101 (1983). d Plasmid commercially available from Pharacia-LKB Biotechnology, Inc. (Piscataway, N J). e j. Vieira and J. Messing, this series, Vol. 153, p. 3. f J. Norrander, J. Vieira, I. Rubinstein, and J. Messing, J. Biotechnol. 2, 157 (1985). g J. M. Short, J. M. Fernandez, J. A. Sorge, and W. D. Huse, Nucleic Acids Res. 16, 7583 (1988). h Plasmid commercially available from Invitrogen Corp. (San Diego, CA). i This paper. J J. Vieira and J. Messing, Gene 100, 189 (1991) k E. Amann and J. Brosius, Gene 40, 183 (1985). t E. Amann, B. Ochs, and K.-J. Abel, Gene 69, 301 (1988). m j. Brosius, Gene 27, 151 (1984).
472
VECTORS FOR CLONING GENES
[41]
A. DraIII PflMI TthlllI BstEII
NcoI
XmnI BsmI
BstBI AIwNI BsgI EcoRI BspMI
AatII SalI
Bsu36I
ID
70
CCATGGCTGGTGACCACGTCGTGGAATGCCTTCGAATTCAGCACCTGCACATGGGACGTCGACCTGAGGT
EcoOl09I ApaI 13A SmaI 4D
13D
BamHI
MunI 15D
(BclI)
BspHI
BglII
(ClaI) BsaBI 10B P v u I BssHII EcoRV
AATTATAACCCGGGCCCTATATATGGATCCAATTGCAATGATCATCATGACAGATCTGCGCGCGATCGAT
DraI IA Eco47III
SphI 3D Fspl N h e I 13B
XbaI
AgeI KpnI
2A HpaI
140
NaeI
SnaBI
ATCAGCGCTTTAAATTTGCGCATGCTAGCTATAGTTCTAGAGGTACCGGTTGTTAACGTTAGCCGGCTAC
BspEI AseI Bstl107I SspI
AvrII StuI
NdeI NsiI
EagI NotI
NarI PvuII PstI KasI
210
BsiWI MluI
ClaI
GTATACTCCGGAATATTAATAGGCCTAGGATGCATATGGCGGCCGCCTGCAGCTGGCGCCATCGATACGC
NruI
SacII
5 A 12D
XhoI SacI
SpeI Scal
BglI SfiI MscI PmlI
ApaLI
280
AflII HindIII
GTACGTCGCGACCGCGGACATGTACAGAGCTCGAGAAGTACTAGTGGCCACGTGGGCCGTGCACCTTAAGCT
T
B. BstBI EcoRI
MscI NcoI
AatII SalI
GAATTCGAATGGCCATGGGACGTCGAC...
FIG. 1. Sequences of superlinkers SL1 and SL2. (A) The coding strand of the 353-bp-long linker SL2 is shown in its entirety. The ATG start codon, which is proximal to an open reading frame that extends through the entire sequence, is underlined. Hexameric and octameric restriction enzyme recognition sites are shown. The 12 palindromic hexameric
[41]
COMPILATION OF SUPERLINKER VECTORS
473
in the recombinant M13 phages, 3'4 to the coding strand with respect to lacZ sequences. Vector pSL300 (Fig. 2), based on pBluescript KS(+),5 as well as pSL350 (Fig. 3) and pSL351, which are derived from pSE1200, 2 have a multitude of features. All harbor superlinker version SL2 (353 bp long; Fig. IA), which is flanked by transcription promoters T3 (proximal to the N c o I site) and T7 (distal to HindIII) for in vitro synthesis ofRNA, pSL300 has the IR from bacteriophage fl, while pSL350 and pSL351 feature the related IR of M 13 phage. In pSL351 the IR is in the alternative orientation. This permits isolation of the opposite DNA strand (the anticoding strand with respect to lacZ sequences), compared to pSL350, which produces the coding single strand on superinfection with helper phage. The yield of single-stranded DNA from pSL351, however, is significantly lower than from pSL350. This is presumably due to interference with replication caused by transcription from a flanking promoter into the phage IR.6 Vieira and Messing 6 therefore recommend conditions for maximal suppression of the wild-type lac promoter during single-stranded DNA preparations, including the use of a lacl q strain and the presence of 0.2-0.4% (w/v) glucose in the growth medium. For sequencing single- or double-stranded DNA the usual two primers flanking the polylinker (e.g., corresponding to T3 and T7 promoters) are not sufficient any more. For most sequence applications, especially when the insert is in central parts of the long superlinker, primers that are located closer to the target of interest are necessary. Therefore, a set of additional primers located at a relatively short distance from any of the cloning sites have been designed and tested for sequencing. 2 In pSL300 and pSL350/1 an attempt was undertaken to maintain, despite the large polylinker, a color discrimination for insert-bearing clones. 5 j. M. Short, J. M. Fernandez, J. A. Sorge, and W. D. Huse, Nucleic Acids Res. 16, 7583 (1988). 6 j. Vieira and J. Messing, Gene 100, 189 (1991).
restriction sites without yet commercially available enzymes are marked by a number and a letter: IA, AAATTT; 1D, TAATTA; 2A, AACGTT, 3D, TAGCTA; 4D, TATATA; 5A, ACATGT; 10B, CGCGCG; 12D, TGTACA; 13A, ATATAT; 13B, CTATAG; 13D, TTATAA; 15D, TTGCAA. The first letter or number of a restriction site designation is located above the 5' nucleotide of its recognition sequence. For clarity, the numerous enzymes with multiple hexameric recognition sequences, or pentameric and tetrameric recognition sequences, have not been included. Sites in brackets are sensitive to methylation by the dam modification system. (B) Sequence variation of the SL1 superlinker. Base 14 and beyond of the SLI linker is identical with base 50 and beyond of SL2 in (A).
474
VECTORS FOR CLONING GENES
[41]
[SacU Eael \ Hindl.
Pvu'ff~ Nsp[~
Banl
PvuI--,
i Asel
W" R
CfrlOI
6,AT
GTA"
T7
/ I000
\
- Asel
Tf"
"EaeI
lac
500
5A CNso HI
IA
pSLSO0
......... 0
1500~
Oril
3 2 6 4 bp
~ BII
2000 \
rNI
Apr
2500 I
Scal
oHI AvaIT
\ CfrlOI ~,I Bqll
15A
Ava]I
FIG. 2. Circular map of pSL300 (drawn to scale). The black heavy segment depicts the SL2 polylinker. The/3-1actamase gene conferring resistance to ampicillin (Ap') and its direction of transcription as well as the plasmid origin (Ori) of replication are shown by arrows. The IR of the fl phage is marked by fl Off. The lac, T3, and T7 promoters and their transcriptional directions are shown by arrows. The ATG translational start codon downstream from Pi, c, the ATG codon at the 5' end of the linker that is part of an NcoI site, the ATG distal to the translational restart (R), as well as the translational stop codon TAA distal to the linker are to be read counterclockwise. Restriction sites in square brackets were formerly bordering the pBluescript KS(+ ) polylinker and are inactivated in this vector. For designations of restriction sites that are not yet commercially available, see the caption to Fig. 1. The numbers of the inner circle give the position on the map in base pairs.
[41]
COMPILATION OF SUPERLINKER VECTORS ,PvuII
-- DrdI A s e I / ~ Rsrl/ ///13A _ /t" / / A s e I
475
,
/
ApaLI
Dri
AIwNI *Eco57I \
\1
~¢
0 500 /
~3000
pSL350 (3594bp) 13A
lacZ 1000---_.
j 2500
*Earn BsaI * Gsul BglI AseI
2000
Apr
1500\
M13 Ori
/
PvuI / ScaI XmnI
BspHI
FIG. 3. Circular map of pSL350 (drawn to scale). The black heavy segment depicts the SL2 polylinker. The fl-lactamase gene conferring resistance to ampicillin (Apt), the lacZ region including the c~ peptide, as well as the IR of the M.13 phage (M13 Ori) correspond to shaded areas. The plasmid origin (Ori) of replication is shown by an open arrow. The lac, T3, and T7 promoters and their transcriptional directions are shown by filled arrowheads. The translational restart between the superlinker (SL) and the lacZ gene segment is marked by an R. For designations of restriction sites that are not yet commercially available, see the caption to Fig. I. Restriction sites with an asterisk are not present in the superlinker. For clarity, only the Ncol and HindlII sites of the polylinker, located at its 5' and 3' ends respectively, are shown. The numbers of the inner circle give the position on the map in base pairs.
Translation should start at the 5' end of the polylinker and proceed through the entire superlinker, which has one open reading frame. Distal to the linker translation stops and restarts into lacZa, the actual indicator gene. This N-terminal portion of fl-galactosidase, if expressed, complements the lacZ mutation of the host. Parent vectors should therefore confer a blue color, while recombinant vectors should be white or light blue. Inserts in the polylinker are expected to contain frequent translational stop codons, thus terminating translation prior to reaching the intergenic region located
476
VECTORS FOR CLONING GENES
[41]
Plac • . . OP. . TTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCA T3 glO CACAGACGAGGATGTCGGACCGAAATTAACCCTCACTAAAC~GGAGATAATTTTGTTTAAC RBS MC RBS NcoI TTTAAGAAGGAGATATATTAATGGCTCGATTAAATAAGGAGGAATAAACCATGGCTGGTG... MetAlaArgLeuAsnLysGluGlu*** M e t A l a G l y ....
superlinker
•G A G A A G T A C T A G T G C . C C A C G T G G G C C G T G C A C C T
SL2
HindIII T7 TAAGCTTCCC TATAGTGAGTCGTAT
T
• . .G l u V a l L e u V a l A l a T h r T r p A l a V a l H i s L e u L y s L e u P r o T y r S e r G l u S e r T y r T
RBS ACAATAAGGAGGAATAACATATGACCATGATTACGGATTCACTGGCCGTCGTTTTACAAC yrAsnLysGluGlu*** MetThrMetIleThrAsDSerLeuAlaValValLeuGlnA
GTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTT rqAraAsDTrDGluAsnPr~GlyValThrGlnL~nAsnArqLeuAlaAlaHisProPr~p
lacZ~
TCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCA heAlaSerTrDAraAsn~er~lu~inAl~AraThrAsDAraProSerGln~inLeuArq~
GCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTT... erLeuAsnGlyGluTrpArgLeuMetArgTyrPheLeuLeu...
FIG. 4. Sequence of relevant area in pSL350/1. Every tenth nucleotide in the first line is marked by a dot. The - 35 and - 10 regions of the lac promoter are underlined. The lac operator (OP) is shown in italics. T3 and T7 transcription promoters are underlined. The bacteriophage T7 gene 10 translaton enhancer (glO) is in italics. Ribosome-binding sites (RBS) preceding the ATG start codons (in italic) of the minicistron (MC), the superpolylinker, and the LacZa peptide are underlined. Stop codons are designated by three asterisks. The deduced amino acid sequences of the minicistron, the linker, and the lacZ region (wild type underlined) are shown. Only the 5' and 3' portions of the superlinker, with flanking NeoI and HindIII sites (underlined), are shown•
between the open reading flame (ORF) of the superlinker and the lacZa coding region. This goal has partially been achieved, first in pSL300, where several test inserts conferred a light-blue colony color versus the midblue color of the nonrecombinant vector.: However, especially when older indicator plates were used it took a day or more for the development of sufficient color. Because the restart occurred into a LacZa' peptide that was still interrupted by T7 promoter sequences, the next series of vectors was designed to restart translation into wild-type lacZa. Therefore, the T7 promoter was moved closer to the 3' end of the polylinker, between HindIII and the translational restart (Fig. 4). On the plates described below, pSL350/1 developed sufficient color in overnight incubations. Color differences between parent and recombinant vectors, however, were
[41]
COMPILATION OF SUPERLINKER VECTORS
477
as subtle as before. For successful use of these vectors in subcloning the following precautions are recommended. I. For subcloning inserts with ends generated by different restriction sites it is important to cut the recipient vector with both enzymes to completion and purify the backbone from the linker fragment. This can easily be achieved by purification on agarose gels and recovery of DNA with, e.g., GlassMilk (BIO I01, La Jolla, CA). 7 No color discrimination is necessary, because the vast majority of clones will represent the desired recombinants. As in all polylinkers it may be difficult to digest two restriction sites that are in close proximity to each other. If such a combination must be used a stuffer fragment should be inserted into site A, resulting in two A sites flanking the insert. Then, after digesting the other site (site B), one of the A sites (the one far from site B) can be digested. 2. For subcloning inserts with identical ends, the linearized vectors can be treated with nuclease-free phosphatase to reduce the background of nonrecombinant vectors. 8 For equal distribution, indicators should be added to the autoclaved medium prior to pouring the plates. Application to the surface leads to uneven distribution of X-Gal, resulting in localized color variation of bacterial colonies even when they contain identical plasmids. 3. It should also be mentioned that not all restriction enzymes are unique in or to the superlinkers. SL2, for example, contains two ClaI sites. However, only one of the ClaI sites is cleavable in normal strains. The second site is overlapped by GATC and is, as the unique BclI site, cleavable only in dam strains. Because BstNI sites are absent in the superlinkers, there is no overlap and no potential inactivation due to methylation of restriction sites by the dcm modification system. 4. Some restriction sites occur once or even several times in other areas of the plasmids (see Figs. 2, 3, 5, and 6). If any of the multiple sites are intended for cloning, a limited digest with the given enzyme is recommended. The superlinkers do not contain the octameric recognition sequences AscI, PacI, PmeI, SrfI, Sse8387I, and SwaI. Likewise, BbsI,
BcgI, Bpull02I, BsaI, BstXI, DrdI, Eamll05I, Earl, Eco57I, EcoNI, Esp3I, GsuI ( = BpmI), PpuMI, RrslI, SgrAI, and XcmI, which are interrupted palindromic or nonpalindromic hexamers, are not in the linker. Some of the above hexameric sites are present in the various vector sequences and can be useful for future plasmid constructions. A version
7 B. Vogelstein and D. Gillespie, Proc. Natl. Acad. Sci. U.S.A. 76, 615 (1979). 8 A. Ullrich, J. Shine, J. Chirgwin, R. Pictet, E. Tisher, W. J. Rutter, and H. M. Goodman, Science 196, 1313 (1977).
478
VECTORS FOR CLONING GENES
[41]
L:~°rtKN NCOl Ase J / ~_.
/Hindlll Xmnl , ~ ~
Asel Aset
5S
AGT
GTA"
,"
T1 i
T2
Sspl
5O0
0//
~'~
1000
EcoRV
• BssHII
Banff 4000-
Pvul ~
I
Apr
_,oo
4467bp
- BstXI -
Bcll
-Mlul ApaLI 3500
• 2000
-BstXl
CfrlOr CfrlOI
3000 2500 i
SphU 0B
Ori
10B
BspHI
AIwNI
~l/ / \ Acci ApaLI
5A
ApaU Rsal
FIG. 5. Circular map of expression vector pSE380 (drawn to scale), pBR322 derived sequences are stippled. The black heavy segment depicts the SL2 polylinker downstream from the trc promoter (arrow), the lacZ translation initiation region including the ribosomebinding site (RBS), and the ATG start codon within the NcoI site. The polylinker is located upstream from a TGA stop codon, the rrnB 5S rRNA gene (cross-hatched) and the rrnB double transcription terminators T1 and T2. The/3-1actamase gene conferring resistance to ampicillin (Apt), the lac repressor gene (laclq) in their directions of transcription, as well as the plasmid origin (Ori) of replication are shown by arrows. An additional HpaI restriction site has been located at the 3' end of the lacl gene by restriction mapping (C. Conlin, J. Zhao, personal communications). A likely position of the site is near bp 4203 (see arrowhead), where a single C-T transition would change an HinclI site into an HpaI site. The numbers of the inner circle give the position on the map in base pairs.
[41]
COMPILATION OF SUPERLINKER VECTORS
479
/ npaI~
~
I
/
/
/~/ClaI coI
EeoRV BssHII
ApaI ~ BstEII -
NsiI N
/ -
~
rtrc / ~
~
.
~.~
SphI --~
--~
/~SL
at" giO \ "~L--
pSE420
Iooo~
(4617bD)
TI_
1 ]
]'2
BspHI
BssHII
*DrdI ~ Bst1107I -~ NdeI /
/3000
1500N 2500 /
~
Ori
2000 \
~ XmnI NDraI
Ap ~
ScaI
DrdI AlwNI FIG. 6. Circular map of expression vector pSE420 (drawn to scale). The black heavy segment depicts the SL2 polylinker. The fl-lactamase gene conferring resistance to ampicillin (Apt), the lac repressor gene (lacIq), and the rrnB 5S ribosomal RNA gene correspond to shaded areas. The plasmid origin (Ori) of replication is shown by an open arrow. The trc promoter (arrowhead) is followed by the rrnB antitermination region (at), the translational initiation region including the translational enhancer from T7 gene 10 (glO), and a minicistron (MC) upstream from the ATG-containing NcoI site. For clarity, the following sites that are both in the superlinker and the remainder of the vector have been omitted (in brackets are the number of sites found outside of the linker): ApaLI (5), AseI (5), BsgI (4), PflMI (1), TthlllI (1), 2A (4), 4D (2), 5A (1), 10B (4), 13A (2), and 15D (1). Also not shown are the following sites that are not in the linker, but in the vector: BbsI (2), BcgI (1), BsaI (2), BstXI (3), Eamll051 (1), Earl (3), Eco57I (2), GsuI (2), and Xcml (3). The two Drdl sites (not present in the superlinker) are marked with an asterisk. Not present in the entire plasmid are AscI, Bpull02I, EcoNI, Esp31, PacI, PmeI, PpuMI, RsrlI, SgrAI, SrfI, Sse83871, and SwaI. For clarity, only the NcoI and HindIII sites of the polylinker, located at its 5' and 3' ends, respectively, are shown. The numbers of the inner circle give the position on the map in base pairs.
480
VECTORS FOR CLONING GENES
[41]
of pSL350, with a shorter polylinker from pTrc99A, 9 yielding pKK489-7, is also available to facilitate future plasmid modifications involving certain restriction sites. Thereafter, the long SL2 superlinker can be reincorporated. 5. Plasmid pSL300, and especially pSL350/1, also have the potential to double as expression vectors. With pSL300 only fusion proteins with N-terminal extensions can be generated. In contrast, pSL350/1 can direct overproduction of unfused proteins when the coding regions are ligated to the unique N c o I site that includes the ATG start codon at the 5' end of the polylinker. This ATG start codon is preceded by regulatory elements for efficient translation initiation, including an upstream minicistron in pSL350/1 (Fig. 4). However, the potential to express a protein encoded by an inserted fragment could also lead to cloning difficulties: If the expressed protein is toxic to the cell, certain inserts may be selected against. If such a scenario is suspected, cloning of a given fragment still could be achieved (without color selection) by down regulating the lac promoter with glucose in a lacI q strain (see above). Expression Vectors Plasmids pSE220, pSE280, pSE380, and pSE420 were designed exclusively as expression vectors for the highly efficient production of recombinant proteins in Escherichia coli. All vectors feature the strong trp/lac fusion promoter trep that can be repressed by cellular Lac repressor and induced with IPTG. l° The difference between pSE220 and its parent pKK233-2,11 which so far has been used in the overproduction of well over 100 different proteins, is the larger polylinker SL2. Expression vectors in this summary feature an ATG start codon within a unique N c o I site located at the 5' end of the superlinker at the appropriate distance from regulatory elements for translation initiation. For expressing unfused proteins this ATG will be the first choice for joining the 5' end of the gene to be overexpressed. All the remaining restriction sites are useful to accommodate ahnost any combination 3' to the protein-coding region for cloning in the appropriate orientation. A shorter version, pSE280, has been generated by eliminating the inactive tet gene of pSE220, pSE380 carries its own gene for the Lac repressor (laclq). Consequently, any E. coil strain with features that may be advantageous for protein overexpression (e.g., protease deficiency) can be used as host. While pSE220, pSE280, and pSE380 contain only the 9 E. A m a n n , B. Ochs, and K.-J. Abel, Gene 69, 301 (1988). to j. Brosius, M. Erfle, and J. Storella, J. Biol. Chem. 260, 3539 (1985). tl E. A m a n n a n d J. Brosius, Gene 40, 183 (1985).
[41]
481
C O M P I L A T I O N OF S U P E R L I N K E R VECTORS Ptrc • OP. TTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCAC r r n B antiterm ACAGGAAACAGCGCCGCTGAGAAAAAGCGAAGCGGCACTGCTCTTTAACAATTTATCAGA glO CAATCTGTGTGGGCACTCGACCGGAATTGGGCATCGATTAACTTTATTATTAAAAATTAA
RBS MC RBS NCOI AGAGGTATATATTAATGTATCGATTAAATAAGGAGGAATAAACCATGGCTGGTGACCACG MetTyrArgLeuAsnLysGluGlu*** MetAlaGlyAspHisV
TCGTGGAATGCCTTCGAATTCAGCACCTGCACATGGGA... alValGluCysLeuArgIleGlnHisLeuHisMetGly...
superlinker
SL2
FIG. 7. Sequence of relevant area in pSE420. Every tenth nucleotide in the first line is marked by a dot. The - 35 and - 10 regions of the trc promoter are underlined. The lac operator (OP), the rrnB antiterminator region, and the bacteriophage T7 gene 10 translation enhancer (glO) are shown in italics. Ribosome-binding sites (RBS) preceding the ATG start codons (in italic) of the minicistron (MC) and the superpolylinker are underlined. Stop codons are designated by three asterisks. The deduced amino acid sequences of the minicistron and the linker are shown. Only the 5' portion of the superlinker region is shown. The N c o I cloning site is underlined.
ribosome-binding site (Shine-Dalgarno sequence) (RBS) from lacZ, pSE420 contains signals of translation initiation that separately have been proven to be highly efficient. Therefore, in addition to all the characteristics of pSE380, the further improved vector pSE420 contains the bacteriophage T7 gene 10 translation enhancer, 12 an upstream minicistron for efficient translational restart,13 and ribosome-binding sites that are flanked by A/T-rich regions 14(see Fig. 7). Furthermore, in pSE420 the transcriptional antitermination region from the E coli rrnB ribosomal RNA operon 15 has been placed downstream from the trc promoter. This may facilitate transcription through highly structured areas of the recombinant messenger RNA and thus reduce the possibility of pausing and/or premature termination of transcription by the host RNA polymerase. As with the majority of expression systems, total repression of promoter activity cannot be achieved in the vectors summarized here. Occasionally this may lead to problems when even small levels of the recombinant protein are toxic to the host cell. I-~p. O. Olins, C. S. Devine, S. H. Rangwala, and K. S. Kavka, Gene 73, 227 (1988). ~3 B. Schoner, R. M. Belagaje, and R. G. Schoner, Proc. Natl. Acad. Sci. U.S.A. 83, 8506 (1986). t4 M. K. Olsen, S. K. Rockenbach, K. A. Curry, and C.-S. C. Tomich, J. Biotechnol. 9, 179 (1989). i5 j. Brosius, T. J. Dull, D. D. Sleeter, and H. F. Noller, J. Mol. Biol. 148, 107 (1981).
482
VECTORS FOR CLONING GENES
[41]
When pSE420 has been tested to direct synthesis of rat calmodulin cDNA, t6 yielding pKK480-3, a large percentage of the cellular protein corresponded to the overproduced species. This vector can serve as a control to monitor the appropriate induction and other parameters in gene expression studies. Of course, some of the parameters, e.g., growth medium, length of induction, or temperature after induction, ~7 may have to be adjusted from case to case to achieve optimal yields or to maximize the fraction of protein with the native conformation and activity. Promoter Selection Vectors pKK232-8, a promoter cloning and selection vector, m has been upgraded with superlinker SL2 yielding pKK520-3. This plasmid contains a promoter-deficient chloramphenicol acetyltransferase gene (cat). The reporter gene is flanked by rrnB transcription terminators to isolate cat from plasmid-borne promoters for reduction of background. The polylinker for inserting or shotgunning promoter fragments is proximal to the cat reporter gene. Cloning of promoter-containing fragments will confer chloramphenicol resistance to the vector and relative levels of CAT can be assayed. ~9Due to the transcriptional isolation of the cloning region this type of vector has also proven to be useful in cloning strong promoters, that otherwise would interfere with plasmid functions. Materials and Reagents Most of the methods for the use of these vectors are sufficiently described elsewhere and in earlier volumes of this series. References are given in the text above. The "Geneclean" kit from BIO 101 (LaJolla, CA) has been used as a source of GlassMilk and buffers to purify DNA from agarose gels for cloning purposes. Bacteria XL 1-Blue is the E. coli strain that has been used for the cloning vectors: recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F'proAB laclqZAM15, Tnl0(Tetr)].20 t6 A. A. Sherbany, A. S. Parent, and J. Brosius, DNA 6, 267 (1987). t7 L. H. Chen, P. C. Babbitt, J. R. V~isquez, B. L. West, and G. L. Kenyon, J. Biol. Chem. 266, 12053 (1991). 18 j. Brosius, Gene 27, 151 (1984). ~9j. Brosius and J. R. Lupski, this series, Vol. 153, p. 54. 20 W. O. Bullock, J. M. Fernandez, and J. M. Short, BioTechniques 5, 376 (1987).
[42]
pBLUESCRIPT II
483
Indicator Plates for Color Screening of Insert Containing Clones Plates are LB (Luria-Bertani) containing 100 /~g/ml ampicillin, 80 /zg/ml X-Gal, and 30 mM IPTG: Per liter, 15 g Bacto-agar, 10 g Bactotryptone, 5 g yeast extract, and 5 g NaCI are autoclaved. After cooling to about 50° the following solutions are added under swirling: l0 ml ofampicillin (10 mg/ml), 3 ml 100 mM IPTG, and 80 mg of X-Gal freshly dissolved in 2 ml dimethylformamide (DMF). Use glass pipettes but not polystyrene pipettes to dispense DMF, because the plastic material is not resistant against the organic solvent.
[42] p B l u e s c r i p t I I : M u l t i f u n c t i o n a l C l o n i n g and Mapping Vectors
By MICHELLE A. ALTING-MEES,J. A. SORGE,and J. M. SHORT pBluescriptII (Stratagene, La Jolla, CA) phagemid vectors are multifunctional cloning vectors designed to simplify and expedite gene cloning and analysis. The vectors contain polylinkers that have been designed to optimize a number of cloning procedures including directional cloning, gene mapping, unidirectional deletions of predictable length, T3 and T7 polymerase-mediated transcription, blue/white color selection for clones with insert, double-stranded DNA sequencing, and fl-galactosidase fusion protein expression. The fl filamentous phage origin of replication allows preparation of single-stranded DNA for mutagenesis and DNA sequencing. The ColE1 origin of replication and the ampicillin resistance gene allow propagation and selection of clones in plasmid form.
Introduction to pBluescript Vectors A map of the pBluescript vectors is given in Fig. 1A. The inset defines the orientations of the fl origin in the plus ( + ) and minus ( - ) versions. The polylinkers are shown for pBluescriptII S K ( + / - ) , pBluescriptII K S ( + / - ) , and the original version pBluescriptI S K ( + / - ) . Figure IB gives the sequence and restriction sites of the pBluescriptII SK polylinker. These polylinkers are located within the a-complementing portion of the /3-galactosidase (lacZ) gene and allow blue/white color selection of clones containing inserts when used in Escherichia coli strains containing the METHODS IN ENZYMOLOGY, VOL. 216
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
COMPILATION OF SUPERLINKER VECTORS
469
[4 1] C o m p i l a t i o n o f S u p e r l i n k e r V e c t o r s
By J~3R~EN BROSIUS There are 64 possibilities for uninterrupted palindromic hexameric restriction enzyme recognition sequences. Enzymes for 52 of those sequences are commercially available. 1Many of the remaining 12 will follow over the next years. A large polylinker has been synthesized that accommodates all the above 64 restriction sites as well as 2 octameric (NotI and Sill) and a few interrupted palindromic and nonpalindromic hexamer sites.2 Several variants of the linker have been inserted into a number of popular multifunctional cloning and expression vectors. 2 These vectors will facilitate many cloning and subcloning applications because of their potential to accommodate restriction fragments irrespective of the enzymes by which they have been generated. In most cases these restriction sites will be preserved. Multifunctional Cloning Vectors Characteristics of some of the more frequently requested as well as four further developed cloning and expression vectors are summarized in Table I. pSL180, pSL190, pSLll80, and pSLI1902 correspond to commonly used vectors pUC18, pUC 19, pUC118, and pUC119. 3 The polylinkers of the latter group have been replaced with the SL1 version [317 base pairs (bp), flanked by EcoRI and HindIII] of the superlinker (Fig. 113). Because of the extended interruption of the LacZa' peptide derivative, a color discrimination between nonrecombinant and insert-containing vectors is not possible anymore. Therefore, bacterial colonies harboring those plasmids are white on indicator plates containing 5-bromo-4-chloro-3-indolyl-fl-D-galactopyranoside (X-Gal) and isopropyl-fl-o-thiogalactopyranoside (IPTG). pSL1180/pSL1190 differ from pSL180/pSL190 by the presence of the intergenic region (IR) from filamentous single-stranded phage M13, which contains the origin of replication as well as signals for phage packaging. This permits the generation of single-stranded DNA following superinfection with helper phage KO7 using a protocol described in an earlier volume of this series. 4 The single-stranded DNA corresponds, as I R. 2 j. 3 j. 4 j.
J. R o b e r t s a n d D. M a c e l i s , Nucleic Acids Res. 20(Suppl.), 2167 (1992). B r o s i u s , D N A 8, 759 (1989). M e s s i n g , Gene 100, 3 (1991). V i e i r a a n d J. M e s s i n g , this series, Vol. 153, p. 3.
METHODS IN ENZYMOLOGY, VOL. 2t6
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
470
VECTORS FOR CLONING GENES
[41]
TABLE I PLASMIDS W I T H SUPERLINKERS a
Size (bp)
Linker
Relevant features
2946
SL1
pSL190 b pUC19 c pSLI180 b'd pUC118 ~
2946 3422
SL1 SL1
pSL1190 b'd pUC119 e pSEI200 b pUC120f
3422 3442
SL1 SL2
pSL300 b
pBS KS(+)g
3264
SL2
pSL301 b,h
pSL300 b
3267
SL2
pSL350 i
pSE1200 b
3594
SL2
pSL351 i
pSL350i/ pUC181 j
3594
SL2
Cloning vector; no color screening for inserts As pSL180; linker in opposite orientation Cloning vector; no color screening for inserts; M 13 origin of replication for generation of single-stranded DNA (the coding strand with respect to the lacZ region) As pSL1180; linker in opposite orientation Cloning and expression vector; no color selection for inserts; M13 origin; lac promoter/operator; lac RBS; ATG start codon in NcoI site SL2 replaces linker from pBluescript KS M13(+); cloning vector; T3 and T7 transcription promoters flanking superlinker; fl origin of replication for generation of single-stranded DNA; translation restart distal to linker into lacZod; subtle color discrimination (midblue to light blue) for insert screening As pSL300; different translation restart from linker into lacZa'; subtle color discrimination (midblue to light blue) for insert screening Cloning and expression vector; T3 and T7 transcription promoters flanking superlinker; M13 origin of replication; lac promoter/operator; T7 gene 10 translational enhancer; minicistron upstream from ATG-containing NcoI site for translational restart; open reading frame throughout superlinker; distal to linker translational restart into wild-type lacZot; subtle color discrimination (blue to midblue) for insert screening As pSL350; M13 origin in opposite orientation; single-stranded DNA (yield significantly lower as with pSL350) representing the anticoding strand with respect to the lacZ region
Plasmid pSL180 b
Parent pUC18 c
[41]
COMPILATION OF SUPERLINKER VECTORS
471
TABLE I (continued)
Plasmid
Parent
Size (bp)
Linker
pKK489-7 ~ pSL350 i
3303
pSE220 b
pKK233-2 k
4926
SL2
pSE280 b,h
pSE220 b
3865
SL2
pSE380 b'h
pTrc99A /
4467
SL2
pSE420 i,h
pSE380 b
4617
SL2
pKK480-3 i
pSE420 i
4760
pKK520-3 i
pKK232-8 m
5417
SL2
Relevant features As pSL350; shorter polylinker (62 bp) from pTrc99AI; useful for future constructions requiring sites not present in the linker Expression vector; trc promoter; lac operator/RBS; inducible with IPTG, ATG in N c o I site; rrnB transcription terminators As pSE220; shorter, remnants of tet gene removed As pSE280; harbors in addition lac repressor (lacI) gene As pSE380; T7 gene 10 translational enhancer; A/T-rich regions flanking RBS; minicistron upstream from CCATGG ( = start codon in N c o I cloning site) for efficient translational restart; open reading frame throughout superlinker As pSE420; rat calmodulin gene instead of linker; model system for very high level of recombinant protein expression Promoter selection vector; linker upstream from promoter-deficient chloramphenicol acetyltransferase gene
All vectors confer resistance to ampicillin. b j. Brosius, D N A 8, 759 (1989). c j. Norrander, T. Kempe, and J. Messing, Gene 26, 101 (1983). d Plasmid commercially available from Pharacia-LKB Biotechnology, Inc. (Piscataway, N J). e j. Vieira and J. Messing, this series, Vol. 153, p. 3. f J. Norrander, J. Vieira, I. Rubinstein, and J. Messing, J. Biotechnol. 2, 157 (1985). g J. M. Short, J. M. Fernandez, J. A. Sorge, and W. D. Huse, Nucleic Acids Res. 16, 7583 (1988). h Plasmid commercially available from Invitrogen Corp. (San Diego, CA). i This paper. J J. Vieira and J. Messing, Gene 100, 189 (1991) k E. Amann and J. Brosius, Gene 40, 183 (1985). t E. Amann, B. Ochs, and K.-J. Abel, Gene 69, 301 (1988). m j. Brosius, Gene 27, 151 (1984).
472
VECTORS FOR CLONING GENES
[41]
A. DraIII PflMI TthlllI BstEII
NcoI
XmnI BsmI
BstBI AIwNI BsgI EcoRI BspMI
AatII SalI
Bsu36I
ID
70
CCATGGCTGGTGACCACGTCGTGGAATGCCTTCGAATTCAGCACCTGCACATGGGACGTCGACCTGAGGT
EcoOl09I ApaI 13A SmaI 4D
13D
BamHI
MunI 15D
(BclI)
BspHI
BglII
(ClaI) BsaBI 10B P v u I BssHII EcoRV
AATTATAACCCGGGCCCTATATATGGATCCAATTGCAATGATCATCATGACAGATCTGCGCGCGATCGAT
DraI IA Eco47III
SphI 3D Fspl N h e I 13B
XbaI
AgeI KpnI
2A HpaI
140
NaeI
SnaBI
ATCAGCGCTTTAAATTTGCGCATGCTAGCTATAGTTCTAGAGGTACCGGTTGTTAACGTTAGCCGGCTAC
BspEI AseI Bstl107I SspI
AvrII StuI
NdeI NsiI
EagI NotI
NarI PvuII PstI KasI
210
BsiWI MluI
ClaI
GTATACTCCGGAATATTAATAGGCCTAGGATGCATATGGCGGCCGCCTGCAGCTGGCGCCATCGATACGC
NruI
SacII
5 A 12D
XhoI SacI
SpeI Scal
BglI SfiI MscI PmlI
ApaLI
280
AflII HindIII
GTACGTCGCGACCGCGGACATGTACAGAGCTCGAGAAGTACTAGTGGCCACGTGGGCCGTGCACCTTAAGCT
T
B. BstBI EcoRI
MscI NcoI
AatII SalI
GAATTCGAATGGCCATGGGACGTCGAC...
FIG. 1. Sequences of superlinkers SL1 and SL2. (A) The coding strand of the 353-bp-long linker SL2 is shown in its entirety. The ATG start codon, which is proximal to an open reading frame that extends through the entire sequence, is underlined. Hexameric and octameric restriction enzyme recognition sites are shown. The 12 palindromic hexameric
[41]
COMPILATION OF SUPERLINKER VECTORS
473
in the recombinant M13 phages, 3'4 to the coding strand with respect to lacZ sequences. Vector pSL300 (Fig. 2), based on pBluescript KS(+),5 as well as pSL350 (Fig. 3) and pSL351, which are derived from pSE1200, 2 have a multitude of features. All harbor superlinker version SL2 (353 bp long; Fig. IA), which is flanked by transcription promoters T3 (proximal to the N c o I site) and T7 (distal to HindIII) for in vitro synthesis ofRNA, pSL300 has the IR from bacteriophage fl, while pSL350 and pSL351 feature the related IR of M 13 phage. In pSL351 the IR is in the alternative orientation. This permits isolation of the opposite DNA strand (the anticoding strand with respect to lacZ sequences), compared to pSL350, which produces the coding single strand on superinfection with helper phage. The yield of single-stranded DNA from pSL351, however, is significantly lower than from pSL350. This is presumably due to interference with replication caused by transcription from a flanking promoter into the phage IR.6 Vieira and Messing 6 therefore recommend conditions for maximal suppression of the wild-type lac promoter during single-stranded DNA preparations, including the use of a lacl q strain and the presence of 0.2-0.4% (w/v) glucose in the growth medium. For sequencing single- or double-stranded DNA the usual two primers flanking the polylinker (e.g., corresponding to T3 and T7 promoters) are not sufficient any more. For most sequence applications, especially when the insert is in central parts of the long superlinker, primers that are located closer to the target of interest are necessary. Therefore, a set of additional primers located at a relatively short distance from any of the cloning sites have been designed and tested for sequencing. 2 In pSL300 and pSL350/1 an attempt was undertaken to maintain, despite the large polylinker, a color discrimination for insert-bearing clones. 5 j. M. Short, J. M. Fernandez, J. A. Sorge, and W. D. Huse, Nucleic Acids Res. 16, 7583 (1988). 6 j. Vieira and J. Messing, Gene 100, 189 (1991).
restriction sites without yet commercially available enzymes are marked by a number and a letter: IA, AAATTT; 1D, TAATTA; 2A, AACGTT, 3D, TAGCTA; 4D, TATATA; 5A, ACATGT; 10B, CGCGCG; 12D, TGTACA; 13A, ATATAT; 13B, CTATAG; 13D, TTATAA; 15D, TTGCAA. The first letter or number of a restriction site designation is located above the 5' nucleotide of its recognition sequence. For clarity, the numerous enzymes with multiple hexameric recognition sequences, or pentameric and tetrameric recognition sequences, have not been included. Sites in brackets are sensitive to methylation by the dam modification system. (B) Sequence variation of the SL1 superlinker. Base 14 and beyond of the SLI linker is identical with base 50 and beyond of SL2 in (A).
474
VECTORS FOR CLONING GENES
[41]
[SacU Eael \ Hindl.
Pvu'ff~ Nsp[~
Banl
PvuI--,
i Asel
W" R
CfrlOI
6,AT
GTA"
T7
/ I000
\
- Asel
Tf"
"EaeI
lac
500
5A CNso HI
IA
pSLSO0
......... 0
1500~
Oril
3 2 6 4 bp
~ BII
2000 \
rNI
Apr
2500 I
Scal
oHI AvaIT
\ CfrlOI ~,I Bqll
15A
Ava]I
FIG. 2. Circular map of pSL300 (drawn to scale). The black heavy segment depicts the SL2 polylinker. The/3-1actamase gene conferring resistance to ampicillin (Ap') and its direction of transcription as well as the plasmid origin (Ori) of replication are shown by arrows. The IR of the fl phage is marked by fl Off. The lac, T3, and T7 promoters and their transcriptional directions are shown by arrows. The ATG translational start codon downstream from Pi, c, the ATG codon at the 5' end of the linker that is part of an NcoI site, the ATG distal to the translational restart (R), as well as the translational stop codon TAA distal to the linker are to be read counterclockwise. Restriction sites in square brackets were formerly bordering the pBluescript KS(+ ) polylinker and are inactivated in this vector. For designations of restriction sites that are not yet commercially available, see the caption to Fig. 1. The numbers of the inner circle give the position on the map in base pairs.
[41]
COMPILATION OF SUPERLINKER VECTORS ,PvuII
-- DrdI A s e I / ~ Rsrl/ ///13A _ /t" / / A s e I
475
,
/
ApaLI
Dri
AIwNI *Eco57I \
\1
~¢
0 500 /
~3000
pSL350 (3594bp) 13A
lacZ 1000---_.
j 2500
*Earn BsaI * Gsul BglI AseI
2000
Apr
1500\
M13 Ori
/
PvuI / ScaI XmnI
BspHI
FIG. 3. Circular map of pSL350 (drawn to scale). The black heavy segment depicts the SL2 polylinker. The fl-lactamase gene conferring resistance to ampicillin (Apt), the lacZ region including the c~ peptide, as well as the IR of the M.13 phage (M13 Ori) correspond to shaded areas. The plasmid origin (Ori) of replication is shown by an open arrow. The lac, T3, and T7 promoters and their transcriptional directions are shown by filled arrowheads. The translational restart between the superlinker (SL) and the lacZ gene segment is marked by an R. For designations of restriction sites that are not yet commercially available, see the caption to Fig. I. Restriction sites with an asterisk are not present in the superlinker. For clarity, only the Ncol and HindlII sites of the polylinker, located at its 5' and 3' ends respectively, are shown. The numbers of the inner circle give the position on the map in base pairs.
Translation should start at the 5' end of the polylinker and proceed through the entire superlinker, which has one open reading frame. Distal to the linker translation stops and restarts into lacZa, the actual indicator gene. This N-terminal portion of fl-galactosidase, if expressed, complements the lacZ mutation of the host. Parent vectors should therefore confer a blue color, while recombinant vectors should be white or light blue. Inserts in the polylinker are expected to contain frequent translational stop codons, thus terminating translation prior to reaching the intergenic region located
476
VECTORS FOR CLONING GENES
[41]
Plac • . . OP. . TTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCA T3 glO CACAGACGAGGATGTCGGACCGAAATTAACCCTCACTAAAC~GGAGATAATTTTGTTTAAC RBS MC RBS NcoI TTTAAGAAGGAGATATATTAATGGCTCGATTAAATAAGGAGGAATAAACCATGGCTGGTG... MetAlaArgLeuAsnLysGluGlu*** M e t A l a G l y ....
superlinker
•G A G A A G T A C T A G T G C . C C A C G T G G G C C G T G C A C C T
SL2
HindIII T7 TAAGCTTCCC TATAGTGAGTCGTAT
T
• . .G l u V a l L e u V a l A l a T h r T r p A l a V a l H i s L e u L y s L e u P r o T y r S e r G l u S e r T y r T
RBS ACAATAAGGAGGAATAACATATGACCATGATTACGGATTCACTGGCCGTCGTTTTACAAC yrAsnLysGluGlu*** MetThrMetIleThrAsDSerLeuAlaValValLeuGlnA
GTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTT rqAraAsDTrDGluAsnPr~GlyValThrGlnL~nAsnArqLeuAlaAlaHisProPr~p
lacZ~
TCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCA heAlaSerTrDAraAsn~er~lu~inAl~AraThrAsDAraProSerGln~inLeuArq~
GCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTT... erLeuAsnGlyGluTrpArgLeuMetArgTyrPheLeuLeu...
FIG. 4. Sequence of relevant area in pSL350/1. Every tenth nucleotide in the first line is marked by a dot. The - 35 and - 10 regions of the lac promoter are underlined. The lac operator (OP) is shown in italics. T3 and T7 transcription promoters are underlined. The bacteriophage T7 gene 10 translaton enhancer (glO) is in italics. Ribosome-binding sites (RBS) preceding the ATG start codons (in italic) of the minicistron (MC), the superpolylinker, and the LacZa peptide are underlined. Stop codons are designated by three asterisks. The deduced amino acid sequences of the minicistron, the linker, and the lacZ region (wild type underlined) are shown. Only the 5' and 3' portions of the superlinker, with flanking NeoI and HindIII sites (underlined), are shown•
between the open reading flame (ORF) of the superlinker and the lacZa coding region. This goal has partially been achieved, first in pSL300, where several test inserts conferred a light-blue colony color versus the midblue color of the nonrecombinant vector.: However, especially when older indicator plates were used it took a day or more for the development of sufficient color. Because the restart occurred into a LacZa' peptide that was still interrupted by T7 promoter sequences, the next series of vectors was designed to restart translation into wild-type lacZa. Therefore, the T7 promoter was moved closer to the 3' end of the polylinker, between HindIII and the translational restart (Fig. 4). On the plates described below, pSL350/1 developed sufficient color in overnight incubations. Color differences between parent and recombinant vectors, however, were
[41]
COMPILATION OF SUPERLINKER VECTORS
477
as subtle as before. For successful use of these vectors in subcloning the following precautions are recommended. I. For subcloning inserts with ends generated by different restriction sites it is important to cut the recipient vector with both enzymes to completion and purify the backbone from the linker fragment. This can easily be achieved by purification on agarose gels and recovery of DNA with, e.g., GlassMilk (BIO I01, La Jolla, CA). 7 No color discrimination is necessary, because the vast majority of clones will represent the desired recombinants. As in all polylinkers it may be difficult to digest two restriction sites that are in close proximity to each other. If such a combination must be used a stuffer fragment should be inserted into site A, resulting in two A sites flanking the insert. Then, after digesting the other site (site B), one of the A sites (the one far from site B) can be digested. 2. For subcloning inserts with identical ends, the linearized vectors can be treated with nuclease-free phosphatase to reduce the background of nonrecombinant vectors. 8 For equal distribution, indicators should be added to the autoclaved medium prior to pouring the plates. Application to the surface leads to uneven distribution of X-Gal, resulting in localized color variation of bacterial colonies even when they contain identical plasmids. 3. It should also be mentioned that not all restriction enzymes are unique in or to the superlinkers. SL2, for example, contains two ClaI sites. However, only one of the ClaI sites is cleavable in normal strains. The second site is overlapped by GATC and is, as the unique BclI site, cleavable only in dam strains. Because BstNI sites are absent in the superlinkers, there is no overlap and no potential inactivation due to methylation of restriction sites by the dcm modification system. 4. Some restriction sites occur once or even several times in other areas of the plasmids (see Figs. 2, 3, 5, and 6). If any of the multiple sites are intended for cloning, a limited digest with the given enzyme is recommended. The superlinkers do not contain the octameric recognition sequences AscI, PacI, PmeI, SrfI, Sse8387I, and SwaI. Likewise, BbsI,
BcgI, Bpull02I, BsaI, BstXI, DrdI, Eamll05I, Earl, Eco57I, EcoNI, Esp3I, GsuI ( = BpmI), PpuMI, RrslI, SgrAI, and XcmI, which are interrupted palindromic or nonpalindromic hexamers, are not in the linker. Some of the above hexameric sites are present in the various vector sequences and can be useful for future plasmid constructions. A version
7 B. Vogelstein and D. Gillespie, Proc. Natl. Acad. Sci. U.S.A. 76, 615 (1979). 8 A. Ullrich, J. Shine, J. Chirgwin, R. Pictet, E. Tisher, W. J. Rutter, and H. M. Goodman, Science 196, 1313 (1977).
478
VECTORS FOR CLONING GENES
[41]
L:~°rtKN NCOl Ase J / ~_.
/Hindlll Xmnl , ~ ~
Asel Aset
5S
AGT
GTA"
,"
T1 i
T2
Sspl
5O0
0//
~'~
1000
EcoRV
• BssHII
Banff 4000-
Pvul ~
I
Apr
_,oo
4467bp
- BstXI -
Bcll
-Mlul ApaLI 3500
• 2000
-BstXl
CfrlOr CfrlOI
3000 2500 i
SphU 0B
Ori
10B
BspHI
AIwNI
~l/ / \ Acci ApaLI
5A
ApaU Rsal
FIG. 5. Circular map of expression vector pSE380 (drawn to scale), pBR322 derived sequences are stippled. The black heavy segment depicts the SL2 polylinker downstream from the trc promoter (arrow), the lacZ translation initiation region including the ribosomebinding site (RBS), and the ATG start codon within the NcoI site. The polylinker is located upstream from a TGA stop codon, the rrnB 5S rRNA gene (cross-hatched) and the rrnB double transcription terminators T1 and T2. The/3-1actamase gene conferring resistance to ampicillin (Apt), the lac repressor gene (laclq) in their directions of transcription, as well as the plasmid origin (Ori) of replication are shown by arrows. An additional HpaI restriction site has been located at the 3' end of the lacl gene by restriction mapping (C. Conlin, J. Zhao, personal communications). A likely position of the site is near bp 4203 (see arrowhead), where a single C-T transition would change an HinclI site into an HpaI site. The numbers of the inner circle give the position on the map in base pairs.
[41]
COMPILATION OF SUPERLINKER VECTORS
479
/ npaI~
~
I
/
/
/~/ClaI coI
EeoRV BssHII
ApaI ~ BstEII -
NsiI N
/ -
~
rtrc / ~
~
.
~.~
SphI --~
--~
/~SL
at" giO \ "~L--
pSE420
Iooo~
(4617bD)
TI_
1 ]
]'2
BspHI
BssHII
*DrdI ~ Bst1107I -~ NdeI /
/3000
1500N 2500 /
~
Ori
2000 \
~ XmnI NDraI
Ap ~
ScaI
DrdI AlwNI FIG. 6. Circular map of expression vector pSE420 (drawn to scale). The black heavy segment depicts the SL2 polylinker. The fl-lactamase gene conferring resistance to ampicillin (Apt), the lac repressor gene (lacIq), and the rrnB 5S ribosomal RNA gene correspond to shaded areas. The plasmid origin (Ori) of replication is shown by an open arrow. The trc promoter (arrowhead) is followed by the rrnB antitermination region (at), the translational initiation region including the translational enhancer from T7 gene 10 (glO), and a minicistron (MC) upstream from the ATG-containing NcoI site. For clarity, the following sites that are both in the superlinker and the remainder of the vector have been omitted (in brackets are the number of sites found outside of the linker): ApaLI (5), AseI (5), BsgI (4), PflMI (1), TthlllI (1), 2A (4), 4D (2), 5A (1), 10B (4), 13A (2), and 15D (1). Also not shown are the following sites that are not in the linker, but in the vector: BbsI (2), BcgI (1), BsaI (2), BstXI (3), Eamll051 (1), Earl (3), Eco57I (2), GsuI (2), and Xcml (3). The two Drdl sites (not present in the superlinker) are marked with an asterisk. Not present in the entire plasmid are AscI, Bpull02I, EcoNI, Esp31, PacI, PmeI, PpuMI, RsrlI, SgrAI, SrfI, Sse83871, and SwaI. For clarity, only the NcoI and HindIII sites of the polylinker, located at its 5' and 3' ends, respectively, are shown. The numbers of the inner circle give the position on the map in base pairs.
480
VECTORS FOR CLONING GENES
[41]
of pSL350, with a shorter polylinker from pTrc99A, 9 yielding pKK489-7, is also available to facilitate future plasmid modifications involving certain restriction sites. Thereafter, the long SL2 superlinker can be reincorporated. 5. Plasmid pSL300, and especially pSL350/1, also have the potential to double as expression vectors. With pSL300 only fusion proteins with N-terminal extensions can be generated. In contrast, pSL350/1 can direct overproduction of unfused proteins when the coding regions are ligated to the unique N c o I site that includes the ATG start codon at the 5' end of the polylinker. This ATG start codon is preceded by regulatory elements for efficient translation initiation, including an upstream minicistron in pSL350/1 (Fig. 4). However, the potential to express a protein encoded by an inserted fragment could also lead to cloning difficulties: If the expressed protein is toxic to the cell, certain inserts may be selected against. If such a scenario is suspected, cloning of a given fragment still could be achieved (without color selection) by down regulating the lac promoter with glucose in a lacI q strain (see above). Expression Vectors Plasmids pSE220, pSE280, pSE380, and pSE420 were designed exclusively as expression vectors for the highly efficient production of recombinant proteins in Escherichia coli. All vectors feature the strong trp/lac fusion promoter trep that can be repressed by cellular Lac repressor and induced with IPTG. l° The difference between pSE220 and its parent pKK233-2,11 which so far has been used in the overproduction of well over 100 different proteins, is the larger polylinker SL2. Expression vectors in this summary feature an ATG start codon within a unique N c o I site located at the 5' end of the superlinker at the appropriate distance from regulatory elements for translation initiation. For expressing unfused proteins this ATG will be the first choice for joining the 5' end of the gene to be overexpressed. All the remaining restriction sites are useful to accommodate ahnost any combination 3' to the protein-coding region for cloning in the appropriate orientation. A shorter version, pSE280, has been generated by eliminating the inactive tet gene of pSE220, pSE380 carries its own gene for the Lac repressor (laclq). Consequently, any E. coil strain with features that may be advantageous for protein overexpression (e.g., protease deficiency) can be used as host. While pSE220, pSE280, and pSE380 contain only the 9 E. A m a n n , B. Ochs, and K.-J. Abel, Gene 69, 301 (1988). to j. Brosius, M. Erfle, and J. Storella, J. Biol. Chem. 260, 3539 (1985). tl E. A m a n n a n d J. Brosius, Gene 40, 183 (1985).
[41]
481
C O M P I L A T I O N OF S U P E R L I N K E R VECTORS Ptrc • OP. TTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCAC r r n B antiterm ACAGGAAACAGCGCCGCTGAGAAAAAGCGAAGCGGCACTGCTCTTTAACAATTTATCAGA glO CAATCTGTGTGGGCACTCGACCGGAATTGGGCATCGATTAACTTTATTATTAAAAATTAA
RBS MC RBS NCOI AGAGGTATATATTAATGTATCGATTAAATAAGGAGGAATAAACCATGGCTGGTGACCACG MetTyrArgLeuAsnLysGluGlu*** MetAlaGlyAspHisV
TCGTGGAATGCCTTCGAATTCAGCACCTGCACATGGGA... alValGluCysLeuArgIleGlnHisLeuHisMetGly...
superlinker
SL2
FIG. 7. Sequence of relevant area in pSE420. Every tenth nucleotide in the first line is marked by a dot. The - 35 and - 10 regions of the trc promoter are underlined. The lac operator (OP), the rrnB antiterminator region, and the bacteriophage T7 gene 10 translation enhancer (glO) are shown in italics. Ribosome-binding sites (RBS) preceding the ATG start codons (in italic) of the minicistron (MC) and the superpolylinker are underlined. Stop codons are designated by three asterisks. The deduced amino acid sequences of the minicistron and the linker are shown. Only the 5' portion of the superlinker region is shown. The N c o I cloning site is underlined.
ribosome-binding site (Shine-Dalgarno sequence) (RBS) from lacZ, pSE420 contains signals of translation initiation that separately have been proven to be highly efficient. Therefore, in addition to all the characteristics of pSE380, the further improved vector pSE420 contains the bacteriophage T7 gene 10 translation enhancer, 12 an upstream minicistron for efficient translational restart,13 and ribosome-binding sites that are flanked by A/T-rich regions 14(see Fig. 7). Furthermore, in pSE420 the transcriptional antitermination region from the E coli rrnB ribosomal RNA operon 15 has been placed downstream from the trc promoter. This may facilitate transcription through highly structured areas of the recombinant messenger RNA and thus reduce the possibility of pausing and/or premature termination of transcription by the host RNA polymerase. As with the majority of expression systems, total repression of promoter activity cannot be achieved in the vectors summarized here. Occasionally this may lead to problems when even small levels of the recombinant protein are toxic to the host cell. I-~p. O. Olins, C. S. Devine, S. H. Rangwala, and K. S. Kavka, Gene 73, 227 (1988). ~3 B. Schoner, R. M. Belagaje, and R. G. Schoner, Proc. Natl. Acad. Sci. U.S.A. 83, 8506 (1986). t4 M. K. Olsen, S. K. Rockenbach, K. A. Curry, and C.-S. C. Tomich, J. Biotechnol. 9, 179 (1989). i5 j. Brosius, T. J. Dull, D. D. Sleeter, and H. F. Noller, J. Mol. Biol. 148, 107 (1981).
482
VECTORS FOR CLONING GENES
[41]
When pSE420 has been tested to direct synthesis of rat calmodulin cDNA, t6 yielding pKK480-3, a large percentage of the cellular protein corresponded to the overproduced species. This vector can serve as a control to monitor the appropriate induction and other parameters in gene expression studies. Of course, some of the parameters, e.g., growth medium, length of induction, or temperature after induction, ~7 may have to be adjusted from case to case to achieve optimal yields or to maximize the fraction of protein with the native conformation and activity. Promoter Selection Vectors pKK232-8, a promoter cloning and selection vector, m has been upgraded with superlinker SL2 yielding pKK520-3. This plasmid contains a promoter-deficient chloramphenicol acetyltransferase gene (cat). The reporter gene is flanked by rrnB transcription terminators to isolate cat from plasmid-borne promoters for reduction of background. The polylinker for inserting or shotgunning promoter fragments is proximal to the cat reporter gene. Cloning of promoter-containing fragments will confer chloramphenicol resistance to the vector and relative levels of CAT can be assayed. ~9Due to the transcriptional isolation of the cloning region this type of vector has also proven to be useful in cloning strong promoters, that otherwise would interfere with plasmid functions. Materials and Reagents Most of the methods for the use of these vectors are sufficiently described elsewhere and in earlier volumes of this series. References are given in the text above. The "Geneclean" kit from BIO 101 (LaJolla, CA) has been used as a source of GlassMilk and buffers to purify DNA from agarose gels for cloning purposes. Bacteria XL 1-Blue is the E. coli strain that has been used for the cloning vectors: recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F'proAB laclqZAM15, Tnl0(Tetr)].20 t6 A. A. Sherbany, A. S. Parent, and J. Brosius, DNA 6, 267 (1987). t7 L. H. Chen, P. C. Babbitt, J. R. V~isquez, B. L. West, and G. L. Kenyon, J. Biol. Chem. 266, 12053 (1991). 18 j. Brosius, Gene 27, 151 (1984). ~9j. Brosius and J. R. Lupski, this series, Vol. 153, p. 54. 20 W. O. Bullock, J. M. Fernandez, and J. M. Short, BioTechniques 5, 376 (1987).
[42]
pBLUESCRIPT II
483
Indicator Plates for Color Screening of Insert Containing Clones Plates are LB (Luria-Bertani) containing 100 /~g/ml ampicillin, 80 /zg/ml X-Gal, and 30 mM IPTG: Per liter, 15 g Bacto-agar, 10 g Bactotryptone, 5 g yeast extract, and 5 g NaCI are autoclaved. After cooling to about 50° the following solutions are added under swirling: l0 ml ofampicillin (10 mg/ml), 3 ml 100 mM IPTG, and 80 mg of X-Gal freshly dissolved in 2 ml dimethylformamide (DMF). Use glass pipettes but not polystyrene pipettes to dispense DMF, because the plastic material is not resistant against the organic solvent.
[42] p B l u e s c r i p t I I : M u l t i f u n c t i o n a l C l o n i n g and Mapping Vectors
By MICHELLE A. ALTING-MEES,J. A. SORGE,and J. M. SHORT pBluescriptII (Stratagene, La Jolla, CA) phagemid vectors are multifunctional cloning vectors designed to simplify and expedite gene cloning and analysis. The vectors contain polylinkers that have been designed to optimize a number of cloning procedures including directional cloning, gene mapping, unidirectional deletions of predictable length, T3 and T7 polymerase-mediated transcription, blue/white color selection for clones with insert, double-stranded DNA sequencing, and fl-galactosidase fusion protein expression. The fl filamentous phage origin of replication allows preparation of single-stranded DNA for mutagenesis and DNA sequencing. The ColE1 origin of replication and the ampicillin resistance gene allow propagation and selection of clones in plasmid form.
Introduction to pBluescript Vectors A map of the pBluescript vectors is given in Fig. 1A. The inset defines the orientations of the fl origin in the plus ( + ) and minus ( - ) versions. The polylinkers are shown for pBluescriptII S K ( + / - ) , pBluescriptII K S ( + / - ) , and the original version pBluescriptI S K ( + / - ) . Figure IB gives the sequence and restriction sites of the pBluescriptII SK polylinker. These polylinkers are located within the a-complementing portion of the /3-galactosidase (lacZ) gene and allow blue/white color selection of clones containing inserts when used in Escherichia coli strains containing the METHODS IN ENZYMOLOGY, VOL. 216
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
