WO1997007220A2 - Linker insertion mutagenesis by means of the transposition system of insertion sequence is21 - Google Patents
Linker insertion mutagenesis by means of the transposition system of insertion sequence is21 Download PDFInfo
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- WO1997007220A2 WO1997007220A2 PCT/EP1996/003625 EP9603625W WO9707220A2 WO 1997007220 A2 WO1997007220 A2 WO 1997007220A2 EP 9603625 W EP9603625 W EP 9603625W WO 9707220 A2 WO9707220 A2 WO 9707220A2
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
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- C12N9/1018—Carboxy- and carbamoyl transferases (2.1.3)
Definitions
- the invention relates to plasmids, corresponding host cells and a genetic engineering method which allows insertions of 4 to 11 codons into cloned genes at any point.
- genes that code for proteins the reading frame is not changed by an insertion. Rather, the inserts modify the protein structure, which means that proteins can receive an altered biological activity.
- the transposition process of the method can take place in vivo, for example in Escherichia coli, or in vitro. The method can also be used to insert epitopes.
- Linker insertion mutagenesis (in-frame insertion mutagenesis; codon cassette mutagenesis) can serve to elucidate structure-function relationships in proteins.
- the insertion of 2, 3, 4 or more codons into genes which code for proteins causes the protein structure to be modified. This can have various consequences: loss of the biological function of the protein, change in the stability or activity of the protein, etc. Under certain circumstances, an insertion does not lead to any detectable change in the target protein (1).
- Previously known methods of linker insertion mutagenesis are mostly based on the insertion of oligonucleotides or selectable cassettes into the target DNA.
- the oligonucleotides or cassettes used generally carry a restriction site.
- Naturally occurring recognition sites for restriction enzymes in the target DNA can be used for linker insertion; in this case the target DNA is opened in vitro with a restriction enzyme (2,3,4).
- the disadvantage of this method is that the insertion sites are not randomly distributed. This limitation can be eliminated with another method: DNase I in lower Concentration and in the presence of Mn 2+ causes double-strand breaks in DNA at any sites (1.5).
- DNase I can often delete several nucleotides near the break site, so that the target DNA along with the desired one Insertion may have further undesired changes (6). This means that many insertion constructs have to be sequenced and that many of them may prove unsuitable.
- IS27 can spontaneously manifest tandem duplications in various bacteria [here designated (IS27) 2 ]. Plasmids with (IS27) 2 form cointegrates together with other plasmids; in E. coli the cointegrate frequency can reach 10 "1 (10, 11). IS27 carries 2 genes, istAB, which are organized as an operon. Their nucleotide sequence is known (12). The istA gene encodes two proteins, which are expressed in the same reading frame and are distinguished by different N-termini: transposase (approx. 46 kDa) and co-integrase (approx. 45 kDa) (10, 11).
- the B gene codes for a protein with an auxiliary function; without this, the transposition frequency is greatly reduced (10, 11.)
- the formation of cointegrates takes place in several steps: first, the (IS27) 2 donor plasmid in the IS27-IS27 connection sequence is opened by cutting at the 3 'ends of the IS elements are set (11). The (IS27) 2 plasmid processed in this way is inserted into the target plasmid by means of a non-replicative mechanism. A target duplication of 4 (rarely 5 ) bp (12). It has now been found that the IS27-IS27 connection sequence can be artificially changed at various points. This creates recognition sites for restriction enzymes without significantly influencing cointegrate formation.
- the present invention thus relates to a method for the random insertion of 4 or 11 codons in any target DNA in a specially constructed mutant of Escherichi coli and with the aid of two plasmids (pME5, pME6).
- the invention thus also relates to a method for the random insertion of 4 or 11 codons into any target DNA in vitro.
- the method is based on three main components (Fig. 1):
- ECOLIST A host strain with chromosomally integrated istA (P45) - and istB genes which are under the control of the tac promoter and the Lacl repressor.
- FIG. 1A Insertions of the "suicide" plasmid into the chromosome of the host strain occur, but are rare.
- Target plasmid and cointegrate can co-exist in the cell for a long time (FIG. 1A).
- the cointegrate is purified by renewed transformation of any strain of E. coli with diluted plasmid DNA (FIG. IB).
- the cointegrates are examined with restriction analysis; Insertions of the "suicide" plasmid in the target gene are examined further, insertions in the vector part are discarded.
- the cointegrates of interest are restricted in vitro with Sall or Bglll. Most of the "suicide" plasmid is lost in the ligation, and an insertion of remains
- the plasmid restricted and ligated with Sall or with BglII is introduced into E. coli by transformation and can be used for the expression of the mutated target gene.
- E. coli ECOLIST is a derivative of the RR28 mutant (14).
- a selectable spectinomycin / streptomycin resistance cassette of pHP45- ⁇ (16) the lad Q gene, the tac promoter (17) and the istA (? 45) - and B gene (13) is inserted into the E. coli ⁇ tt7? locus.
- the expression of the istAB genes cloned under the tac promoter can be induced with isopropyl- ⁇ -D-thiogalactoside (IPTG).
- the "Suicide" plasmids pME5 and pME6 are a derivative of pJP5603 (18), in which a synthetic 0.14 kb oligonucleotide has been inserted as a TfcoRI-7 /.> ⁇ flII fragment.
- the central region is a modified IS27-IS27 connection sequence, which restriction sites for Bgäl and Sall in the "inverted repeats" from IS27 carries (Fig. 2B).
- the plasmid can be replicated in the auxiliary strain E. coli CCI 18 ( ⁇ pir). This strain (19) codes for the protein ⁇ which initiates the replication of the "suicide" plasmid from its oriR ⁇ K. In the absence of the protein ⁇ , this plasmid cannot be replicated.
- the plasmid pME6 (FIG. 2A) is a derivative of pGP704 (19) with a chloramphenicol resistance cassette from Tn7725 (20) and the VBS region (FIG. 2B).
- the principle of protein ⁇ -dependent replication is the same as for pME5.
- the target plasmid pME3659 (Fig. 1) is shown as an example.
- the vector is said to be high
- pBluescript (21) Possess number of copies; pBluescript (21) is suitable.
- the target gene is, for example, the arcB gene from Pseudomonas aeruginosa (22).
- the arcB gene encodes catabolic ornithine carbamoyl transferase (22).
- the culture of this strain is diluted 1: 100 in LB medium (23) and 1 mM IPTG is added. The culture is grown at 37 ° C to OD 550 ⁇ 0.5.
- the cells are transformed with 0.3 ⁇ g pME5 or pME6 (23) and selected on LB plates with 50 ⁇ g canamycin / ml or 50 ⁇ g chloramphenicol / ml. Typically, about 50 colonies resistant to kanamycin or chloramphenicol are obtained; the majority of these contain a cointegrate, in addition to the target plasmid (FIG. 1A).
- Plasmid preparations produced according to the CTAB method (24) are diluted 1: 100 to 1: 1000 and used for the transformation of E. coli ED8767 (25). By selecting for cointegrates (kanamycin or chloramphenicol), these can be purified from the target plasmid. 2.
- cointegrates kanamycin or chloramphenicol
- the method is essentially based on the use of the following 3 components (Fig. 4):
- This plasmid (13) carries Lst.4 (P45) and is the B gene behind the inducible tac promoter.
- the target gene xyz in a high copy number plasmid e.g. pBluescript (Stratagene).
- pME3659 can be used (see in vivo method).
- the cointegration between the target plasmid and pME5 (or pME6) is determined by the im
- the methods according to the invention can be used in particular for the generation, in vivo or in vitro, of new Safl or ßg / II restriction sites in any target genes.
- an oligonucleotide can be used which codes for an immunologically detectable epitope (28) ("epitope mapping").
- epitope (28) immunologically detectable epitope 228
- the 12 bp insertions shown in FIGS. 3A, B were obtained.
- the "suicide" plasmid was pME5 or pME6.
- the position of each insertion was determined by nucleotide sequence analysis. A comparison of the insertion sites obtained showed no homology; there is no evidence that the IS27 transposition system prefers a particular target sequence (12). 45% of the insertion mutants retained enzymatic activity; 55% had lost activity.
- the insertion at position 509 resulted in a catabolic ornithine carbamoyl transferase which could no longer be activated by the allosteric activator AMP (26).
- the crude extract of E. coli ED8767 / pME3913 is produced according to the Schmid method (13).
- the incubation mixture is composed of the following components on ice: pME5 (or pME6) DNA 0.25 ⁇ g 2 ⁇ l
- Target plasmid DNA e.g. pME3659 0.25 ⁇ g 2 ⁇ l
- the reaction mixture is incubated at 30 ° C for 60 minutes. Thereafter, the ratio of cointegrates to target plasmid is approximately IO "3.
- Total plasmid DNA (24) is cleaned once with phenol / chloroform (23) and then with the Geneclean II kit (BIO 101). Cleaned Plasmid DNA is introduced into E. coli ED8767 by electroporation (27), with selection for the kanamycin resistance of pME5 (or the chloramphenicol resistance of pME6).
- Fig. 1 In vivo method; Selection, cleaning and dissolution of coin integrates. The most important restriction interfaces are given.
- the cointegration takes place after the first transformation.
- the target plasmid is also present in cells A.
- After the second transformation with diluted plasmid DNA B contains only one cointegrate.
- SS suicide plasmid, eg pME6; l ] target plasmid, e.g. B. pME3659.
- CM R chloramphenicol resistance.
- Step after B isolation of plasmid DNA, dilution of the DNA IO "2 to IO " 3 and transformation, selection on Cm 50 medium
- Step after C isolation of plamsid DNA, restriction with Safl or BgRl, ligation, transformation
- Fig. 2 VBS plasmids pME5 and pME6.
- A localization of the insertions of pME5 and pME6 in arcB gene.
- the positions refer to the first nucleotide of the ctrC-5 fragment (22).
- the arrows indicate the orientation, which is defined in B. ⁇ ? ⁇ Start of arcB translation; ⁇ End of arcB translation.
- B definition of orientation, after the integration of pME5 and pME6 in arcB ( 1 --- 2 ).
- C sequences of insertions at position 509 and 990. Insertion 509 was made with pME6 and insertion 990 with pME5.
- the orientation of the insertions is defined in B.
- the target duplications are underlined.
- pME5 is shown as a suicide plasmid.
- A The IstA (P45) and IstB proteins required for the reaction are overproduced using a t ⁇ c promoter which has been cloned in front of the IS27 genes. Ap, ampicillin resistance.
- B pME3913 is used for the overproduction of the IS27 proteins. The sequence deviating from the IS27 wild type sequence is given below.
- RBS ribosome binding site *, position on the IS27 sequence (12).
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Abstract
A genetic engineering process allows 4 to 11 codons to be inserted into any site of cloned genes. In genes that code for proteins, the reading pattern is not modified by an insertion. The insertions modify the protein structure, so that the proteins may be given a modified biologic activity. Transposition may be carried out according to this process both in vivo, for example in Escherichia coli, or in vitro. The process may also be used to insert epitopes.
Description
Linker-Insertionsmutagenese mit Hilfe des Transpositioπssystems der Insertionssequenz IS27Linker insertion mutagenesis using the transposition system of the insertion sequence IS27
Die Erfindung betrifft Plasmide, entsprechende Wirtzzellen sowie ein gentechnologisches Verfahren, welches Insertionen von 4 bis 11 Codons in klonierte Gene an beliebiger Stelle erlaubt. In Genen, welche für Proteine codieren, wird der Leseraster durch eine Insertion nicht verändert. Vielmehr modifizieren die Insertionen die Proteinstruktur, wodurch Proteine eine veränderte biologische Aktivität erhalten können. Der Transpositionsvorgang des Verfahrens kann in vivo, beispielsweise in Escherichia coli, oder in vitro stattfinden. Die Methode kann auch zur Insertion von Epitopen dienen.The invention relates to plasmids, corresponding host cells and a genetic engineering method which allows insertions of 4 to 11 codons into cloned genes at any point. In genes that code for proteins, the reading frame is not changed by an insertion. Rather, the inserts modify the protein structure, which means that proteins can receive an altered biological activity. The transposition process of the method can take place in vivo, for example in Escherichia coli, or in vitro. The method can also be used to insert epitopes.
Die Linker-Insertionsmutagenese (linker insertion mutagenesis; in-frame insertion mutage- nesis; codon cassette mutagenesis) kann dazu dienen, Struktur-Funktions-Beziehungen in Pro¬ teinen aufzuklären. Die Insertion von 2, 3, 4 oder mehreren Codons in Gene, welche für Pro¬ teine codieren, bewirkt, daß die Proteinstruktur modifiziert wird. Dies kann verschiedene Konsequenzen haben: Verlust der biologischen Funktion des Proteins, Veränderung der Sta¬ bilität oder der Aktivität des Proteins etc. Unter Umständen führt eine Insertion zu keiner fest¬ stellbaren Veränderung des Zielproteins (1). Bisher bekannte Verfahren der Linker-Inserti¬ onsmutagenese beruhen meist auf dem Einsetzen von Oligonukleotiden oder von selektionier- baren Kassetten in die Ziel-DNA. Die verwendeten Oligonucleotide bzw. Kassetten tragen in der Regel eine Restriktionsstelle. Natürlich vorkommende Erkennungsstellen für Restrikti¬ onsenzyme in der Ziel-DNA können zur Linker-Insertion verwendet werden; in diesem Fall wird die Ziel-DNA in vitro mit einem Restriktionsenzyme an einer Stelle geöffnet (2,3,4). Der Nachteil dieser Methode ist, daß die Insertionsstellen nicht zufällig, verteilt sind. Diese Einschränkung kann mit einer anderen Methode ausgeschaltet werden: DNase I in niedriger
Konzentration und in Anwesenheit von Mn2+ bewirkt Doppelstrang-Brüche in DNA an belie¬ bigen Stellen (1,5) Der Nachteil hier ist, daß DNase I häufig mehrere Nukleotide nahe der Bruchstelle deletieren kann, so daß die Ziel-DNA nebst der erwünschten Insertion noch wei¬ tere unerwünschte Veränderungen aufweisen kann (6). Dies bedeutet, daß viele Insertions- Konstrukte sequenziert werden müssen und daß sich viele von ihnen als ungeeignet Heraus¬ stellen können. Gerichtete In-vitro-Mutagenese mit Oligonukleotiden kann unspezifische Ver¬ änderungen in der Ziel-DNA vermeiden (7,8). Wenn jedoch viele, zufällig verteilte Inser¬ tionen isoliert werden sollen, so wird diese Methode teuer und zeitraubend. Linker-Insertion auf der Basis eines transponierbaren Elementes (Transposon, Insertionssequenz), welches wenig Spezifität bei der Wahl der Ziel-DNA zeigt, kann die oben erwähnten Nachteile ver¬ meiden. Ein solches System ist für ein mini-Tn5-Element beschrieben worden; Insertionen von 135 bp (d.h. 45 Codons) werden damit erhalten (9). So große Insertionen sind aber für die meisten Zwecke ungeeignet, da die Proteinstruktur zu stark unterbrochen wird. Das hier be¬ schriebene Verfahren, welches auf der Insertionssequenz IS27 beruht, hat die erwähnten Nachteile nicht.Linker insertion mutagenesis (in-frame insertion mutagenesis; codon cassette mutagenesis) can serve to elucidate structure-function relationships in proteins. The insertion of 2, 3, 4 or more codons into genes which code for proteins causes the protein structure to be modified. This can have various consequences: loss of the biological function of the protein, change in the stability or activity of the protein, etc. Under certain circumstances, an insertion does not lead to any detectable change in the target protein (1). Previously known methods of linker insertion mutagenesis are mostly based on the insertion of oligonucleotides or selectable cassettes into the target DNA. The oligonucleotides or cassettes used generally carry a restriction site. Naturally occurring recognition sites for restriction enzymes in the target DNA can be used for linker insertion; in this case the target DNA is opened in vitro with a restriction enzyme (2,3,4). The disadvantage of this method is that the insertion sites are not randomly distributed. This limitation can be eliminated with another method: DNase I in lower Concentration and in the presence of Mn 2+ causes double-strand breaks in DNA at any sites (1.5). The disadvantage here is that DNase I can often delete several nucleotides near the break site, so that the target DNA along with the desired one Insertion may have further undesired changes (6). This means that many insertion constructs have to be sequenced and that many of them may prove unsuitable. Directed in vitro mutagenesis with oligonucleotides can avoid unspecific changes in the target DNA (7,8). However, if many, randomly distributed insertions are to be isolated, this method becomes expensive and time-consuming. Linker insertion based on a transposable element (transposon, insertion sequence) which shows little specificity in the choice of the target DNA can avoid the disadvantages mentioned above. Such a system has been described for a mini-Tn5 element; Inserts of 135 bp (ie 45 codons) are thus obtained (9). Such large insertions are unsuitable for most purposes because the protein structure is interrupted too much. The method described here, which is based on the insertion sequence IS27, does not have the disadvantages mentioned.
Die Insertionssequenz IS27 kann spontan in verschiedenen Bakterien Tandemduplikationen [hier mit (IS27)2 bezeichnet] ausprägen. Plasmide mit (IS27)2 bilden mit anderen Plasmiden zusammen Cointegrate; in E. coli kann die Cointegrat-Frequenz 10"1 erreichen (10, 11). IS27 trägt 2 Gene, istAB, welche als Operon organisiert sind. Ihre Nukleotidsequenz ist bekannt (12). Das istA-Gen codiert für zwei Proteine, welche im gleichen Leseraster exprimiert wer¬ den und sich durch verschiedene N-Termini auszeichnen: Transposase (ca. 46 kDa) und Co- integrase (ca. 45 kDa) (10, 11). Das istB-Gen codiert für ein Protein mit Hilfsfunktion; ohne dieses ist die Transpositionsfrequenz stark erniedrigt (10, 11). Die Bildung von Cointegraten verläuft in mehreren Schritten: zuerst wird das (IS27)2-Donorplasmid in der IS27-IS27-Ver- bindungssequenz geöffnet, indem Schnitte an den 3 '-Enden der IS-Elemente gesetzt werden (11). Das solchermaßen prozessierte (IS27)2-Plasmid wird in das Zielplasmid eingesetzt, mit¬ tels eines nicht-replikativen Mechanismus. Durch Reparatur der Ziel-DNA entsteht eine Ziel¬ duplikation von 4 (selten 5) bp (12).
Es wurde nun gefunden, daß die IS27-IS27-Verbindungssequenz an verschiedenen Stellen künstlich verändert werden kann. Dadurch entstehen Erkennungsstellen für Restriktionsen¬ zyme, ohne daß die Cointegrat-Bildung signifikant beeinflußt wird. Die vorliegende Erfin¬ dung betrifft somit ein Verfahren zur zufälligen Insertion von 4 oder 11 Codons in einer be- liebigen Ziel-DNA in einer speziell konstruierten Mutante von Escherichi coli und mit Hilfe von zwei Plasmiden (pME5, pME6).The insertion sequence IS27 can spontaneously manifest tandem duplications in various bacteria [here designated (IS27) 2 ]. Plasmids with (IS27) 2 form cointegrates together with other plasmids; in E. coli the cointegrate frequency can reach 10 "1 (10, 11). IS27 carries 2 genes, istAB, which are organized as an operon. Their nucleotide sequence is known (12). The istA gene encodes two proteins, which are expressed in the same reading frame and are distinguished by different N-termini: transposase (approx. 46 kDa) and co-integrase (approx. 45 kDa) (10, 11). The B gene codes for a protein with an auxiliary function; without this, the transposition frequency is greatly reduced (10, 11.) The formation of cointegrates takes place in several steps: first, the (IS27) 2 donor plasmid in the IS27-IS27 connection sequence is opened by cutting at the 3 'ends of the IS elements are set (11). The (IS27) 2 plasmid processed in this way is inserted into the target plasmid by means of a non-replicative mechanism. A target duplication of 4 (rarely 5 ) bp (12). It has now been found that the IS27-IS27 connection sequence can be artificially changed at various points. This creates recognition sites for restriction enzymes without significantly influencing cointegrate formation. The present invention thus relates to a method for the random insertion of 4 or 11 codons in any target DNA in a specially constructed mutant of Escherichi coli and with the aid of two plasmids (pME5, pME6).
Es wurde darüber hinaus gefunden, daß die Cointegrat-Bildung zwischen pME5 (oder pME6) und einem Zielplasmid in vitro ablaufen kann, in Gegenwart eines Proteinextraktes mit den überexprimierten IstA- und IstB-Proteinen. Die Erfindung betrifft somit zudem ein Verfahren zur zufalligen Insertion von 4 oder 11 Codons in beliebige Ziel-DNA in vitro.It has also been found that cointegrate formation between pME5 (or pME6) and a target plasmid can occur in vitro, in the presence of a protein extract with the overexpressed IstA and IstB proteins. The invention thus also relates to a method for the random insertion of 4 or 11 codons into any target DNA in vitro.
Im folgenden wird sowohl das erfindungsgemäße in-vivo-, als auch das in-vitro-Verfahren im Detail beschrieben.Both the in vivo and the in vitro method according to the invention are described in detail below.
1. In- vivo- Verfahren1. In-vivo procedure
Die Methode beruht auf drei Hauptkomponenten (Fig. 1):The method is based on three main components (Fig. 1):
- Escherichia coli ECOLIST: Ein Wirtsstamm mit chromosomal integrierten istA(P45)- und istB-Genen, die unter der Kontrolle des tac-Promotors und des Lacl-Repressors stehen.- Escherichia coli ECOLIST: A host strain with chromosomally integrated istA (P45) - and istB genes which are under the control of the tac promoter and the Lacl repressor.
- Ein "Suicide"-Plasmid (pME6 oder pME5) mit einer IS27-IS27-Verbindungssequenz (=VBS), welche Restriktionsstellen für die Enzyme Sall und Bgäl enthält (Fig. 2).- A "suicide" plasmid (pME6 or pME5) with an IS27-IS27 connecting sequence (= VBS) which contains restriction sites for the enzymes Sall and Bgal (FIG. 2).
- Ein Ziel-Gen in einem Plasmid mit hoher Kopienzahl. z. B. pME3659 (Fig. 1). Der verwendete Vektor ist z. B. pBluescript (Stratagene).
Der Wirtsstamm ECOLIST wird zuerst mit dem Zielplasmid, dann mit dem "Suicide"- Plasmid transformiert. Cointegrate zwischen den beiden Plasmiden werden selektioniert (Fig. 1 A). Insertionen des "Suicide"-Plasmids ins Chromosom des Wirtsstammes kom¬ men vor, sind aber selten. Zielplasmid und Cointegrat können in der Zelle längere Zeit co-existieren (Fig. 1 A). Durch erneute Transformation eines beliebigen Stammes von E. coli mit verdünnter Plasmid-DNA wird das Cointegrat gereinigt (Fig. IB). Die Cointe¬ grate werden mit Restriktionsanalyse untersucht; Insertionen des "Suicide"-Plasmids im Ziel-Gen werden weiter untersucht, Insertionen im Vektorteil werden verworfen. Die Cointegrate von Interesse werden in vitro mit Sall oder Bglll restringiert. Bei der Ligation geht der größte Teil des "Suicide "-Plasmids verloren, und zurück bleibt eine Insertion von- A target gene in a high copy number plasmid. z. B. pME3659 (Fig. 1). The vector used is e.g. B. pBluescript (Stratagene). The host strain ECOLIST is transformed first with the target plasmid, then with the "suicide" plasmid. Cointegrates between the two plasmids are selected (FIG. 1A). Insertions of the "suicide" plasmid into the chromosome of the host strain occur, but are rare. Target plasmid and cointegrate can co-exist in the cell for a long time (FIG. 1A). The cointegrate is purified by renewed transformation of any strain of E. coli with diluted plasmid DNA (FIG. IB). The cointegrates are examined with restriction analysis; Insertions of the "suicide" plasmid in the target gene are examined further, insertions in the vector part are discarded. The cointegrates of interest are restricted in vitro with Sall or Bglll. Most of the "suicide" plasmid is lost in the ligation, and an insertion of remains
12 bp (8 bp aus der VBS-Region + 4 bp Zielduplikation) bzw. 33 bp (29 bp aus der VBS- Region + 4 bp Zielduplikation) (Fig. IC). Dadurch entstehen im Ziel-Gen Insertionen, welche den Leseraster nicht verändern. Das mit Sall bzw. mit Bglll restringierte und li- gierte Plasmid wird durch Transformation in E. coli eingeführt und kann für die Expres- sion des mutierten Ziel-Gens verwendet werden.12 bp (8 bp from the VBS region + 4 bp target duplication) or 33 bp (29 bp from the VBS region + 4 bp target duplication) (Fig. IC). This creates insertions in the target gene that do not change the reading frame. The plasmid restricted and ligated with Sall or with BglII is introduced into E. coli by transformation and can be used for the expression of the mutated target gene.
Detaillierte Beschreibung der Hauptkomponenten:Detailed description of the main components:
- E. coli ECOLIST ist ein Derivat der Mutante RR28 (14). Mit Hilfe des Klonierungs- vektors pLDRl 0 ( 15) wurden eine selektionierbare Spectinomycin/Streptomycin-Re- sistenzkassette von pHP45-Ω (16), das lad Q-Gen, der tac-Promotor (17) und die istA(?45)- und istB-Gene (13) in den αtt7?-Locus von E. coli inseriert. Mit Isopropyl-ß- D-thiogalactosid (IPTG) kann die Expression der unter dem tac-Promotor klonierten istAB-Gene induziert werden.- E. coli ECOLIST is a derivative of the RR28 mutant (14). With the help of the cloning vector pLDRl 0 (15), a selectable spectinomycin / streptomycin resistance cassette of pHP45-Ω (16), the lad Q gene, the tac promoter (17) and the istA (? 45) - and B gene (13) is inserted into the E. coli αtt7? locus. The expression of the istAB genes cloned under the tac promoter can be induced with isopropyl-β-D-thiogalactoside (IPTG).
Die "Suicide"-Plasmide pME5 und pME6. Das Plasmid pME5 (Fig. 2A) ist ein Deri¬ vat von pJP5603 (18), in welches ein synthetisches 0.14 kb Oligonukleotid als TfcoRI- 7/.>κflII-Fragment inseriert worden ist. Die zentrale Region ist eine veränderte IS27- lS27-Verbindungssequenz, welche Restriktionsstellen für Bgäl und Sall in den
"inverted repeats" von IS27 trägt (Fig. 2B). Das Plasmid kann im Hilfsstamm E. coli CCI 18 (λpir) repliziert werden. Dieser Stamm (19) codiert für das Protein π welches die Replikation des "Suicide"-plasmids von dessen oriRβK aus initiiert. In Abwesen¬ heit des Proteins π kann dieses Plasmid nicht repliziert werden.The "Suicide" plasmids pME5 and pME6. The plasmid pME5 (FIG. 2A) is a derivative of pJP5603 (18), in which a synthetic 0.14 kb oligonucleotide has been inserted as a TfcoRI-7 /.> ΚflII fragment. The central region is a modified IS27-IS27 connection sequence, which restriction sites for Bgäl and Sall in the "inverted repeats" from IS27 carries (Fig. 2B). The plasmid can be replicated in the auxiliary strain E. coli CCI 18 (λpir). This strain (19) codes for the protein π which initiates the replication of the "suicide" plasmid from its oriRβK. In the absence of the protein π, this plasmid cannot be replicated.
Das Plasmid pME6 (Fig. 2A) ist ein Derivat von pGP704 (19) mit einer Chloram- phenicol-Resistenz-Kassette aus Tn7725 (20) und der VBS-Region (Fig. 2B). Das Prinzip der Protein π-abhängigen Replikation ist dasselbe wie bei pME5.The plasmid pME6 (FIG. 2A) is a derivative of pGP704 (19) with a chloramphenicol resistance cassette from Tn7725 (20) and the VBS region (FIG. 2B). The principle of protein π-dependent replication is the same as for pME5.
- Das Ziel-Plasmid pME3659 (Fig. 1) ist gezeigt als Beispiel. Der Vektor soll eine hohe- The target plasmid pME3659 (Fig. 1) is shown as an example. The vector is said to be high
Kopienzahl besitzen; pBluescript (21) ist geeignet. Das Ziel-Gen ist beispielsweise das arcB-Gen von Pseudomonas aeruginosa (22). Das arcB-Gen codiert für katabolische Ornithin-Carbamoyltransferase (22).Possess number of copies; pBluescript (21) is suitable. The target gene is, for example, the arcB gene from Pseudomonas aeruginosa (22). The arcB gene encodes catabolic ornithine carbamoyl transferase (22).
Die Bedingungen für Plasmid-Extraktion, Restriktion, Ligation und Transformation sind gemäß Standard-Methoden (23).The conditions for plasmid extraction, restriction, ligation and transformation are according to standard methods (23).
Nach der Transformation des Stammes ECOLIST mit dem Zielplasmid (z. B. pME3659) wird die Kultur dieses Stammes 1 : 100 in LB-Medium (23) verdünnt und 1 mM IPTG zu- gegeben. Die Kultur wird bei 37 °C bis OD 550 ≡ 0.5 wachsen gelassen. Die Zellen wer¬ den mit 0.3 μg pME5 oder pME6 transformiert (23) und auf LB-Platten mit 50 μg Ka- namycin/ml bzw. 50 μg Chloramphenicol/ml selektioniert. Typischerweise werden so ca. 50 gegen Kanamycin bzw. Chloramphenicol resistente Kolonien erhalten; diese enthalten mehrheitlich ein Cointegrat, nebst dem Zielplasmid (Fig. 1 A). Plasmidpräparate herge- stellt nach der CTAB-Methode (24) werden 1 : 100 bis 1 : 1000 verdünnt und für die Trans¬ formation von E. coli ED8767 (25) verwendet. Durch Selektion für Cointegrate (Kanamycin bzw. Chloramphenicol) können diese vom Zielplasmid gereinigt werden.
2. In-vitro-VerfahrenAfter the transformation of the ECOLIST strain with the target plasmid (eg pME3659), the culture of this strain is diluted 1: 100 in LB medium (23) and 1 mM IPTG is added. The culture is grown at 37 ° C to OD 550 ≡ 0.5. The cells are transformed with 0.3 μg pME5 or pME6 (23) and selected on LB plates with 50 μg canamycin / ml or 50 μg chloramphenicol / ml. Typically, about 50 colonies resistant to kanamycin or chloramphenicol are obtained; the majority of these contain a cointegrate, in addition to the target plasmid (FIG. 1A). Plasmid preparations produced according to the CTAB method (24) are diluted 1: 100 to 1: 1000 and used for the transformation of E. coli ED8767 (25). By selecting for cointegrates (kanamycin or chloramphenicol), these can be purified from the target plasmid. 2. In vitro method
Die Methode beruht im wesentlichen auf der Verwendung folgender 3 Komponenten (Fig. 4):The method is essentially based on the use of the following 3 components (Fig. 4):
- Rohextrakt aus E. coli ED8767 mit dem Plasmid pME3913. Dieses Plasmid (13) trägt die Lst.4(P45)- und istB-Gene hinter dem induzierbaren tac-Promotor.- Crude extract from E. coli ED8767 with the plasmid pME3913. This plasmid (13) carries Lst.4 (P45) and is the B gene behind the inducible tac promoter.
- Das "Suicide"-Plasmid pME5 oder pME6 (siehe In-vivo-Verfahren).- The "Suicide" plasmid pME5 or pME6 (see in vivo method).
- Das Ziel-Gen xyz in einem Plasmid mit hoher Kopienzahl, z.B. pBluescript (Stratagene). Beispielsweise kann pME3659 verwendet werden (siehe In-vivo-Verfah¬ ren).- The target gene xyz in a high copy number plasmid, e.g. pBluescript (Stratagene). For example, pME3659 can be used (see in vivo method).
Die Cointegration zwischen dem Ziel-Plasmid und pME5 (oder pME6) wird durch die imThe cointegration between the target plasmid and pME5 (or pME6) is determined by the im
Rohextrakt vorhandenen IstA(P45)- und IstB-Proteine katalysiert. Cointegrate werden ge¬ reinigt, durch Elektroporation in E. coli ED8767 eingeführt und mit Restriktionsver¬ dauungen analysiert. Insertionen von pME5 (bzw. pME6) im Ziel-Gen xyz werden mit Sall bzw. Bglll ausgeschnitten. Nach Ligation werden Insertionen von 12 bp (für SaR) bzw. 33 bp erhalten (Fig. 4A). Diese sind analog den im In-vivo-Verfahren erhaltenen In¬ sertionen.Crude extract catalyzes existing IstA (P45) and IstB proteins. Cointegrates are cleaned, introduced into E. coli ED8767 by electroporation and analyzed with restriction digestions. Insertions of pME5 (or pME6) in the target gene xyz are cut out with Sall or Bglll. After ligation, inserts of 12 bp (for SaR) or 33 bp are obtained (FIG. 4A). These are analogous to the insertions obtained in the in vivo method.
Die erfindungsgemäßen Verfahren können insbesondere zur Erzeugung, in vivo oder in vitro, von neuen Safl- bzw. ßg/II-Restriktionsstellen in beliebigen Ziel-Genen verwendet werden. An diesen Restriktionsstellen kann beispielsweise ein Oligonukleotid eingesetzt werden, welches für ein immunologisch nachweisbares Epitop (28) codiert ("Epitope mapping").
Die künstlich eingeführten Sall- und ßg/II-Stellen in der VBS-Region (Fig. 2B) sind nicht die einzig möglichen. Wegen der geringen Rekognitionsspezifität der Cointegrase sind weitere neue Restriktionsstellen in den "inverted repeats" möglich.The methods according to the invention can be used in particular for the generation, in vivo or in vitro, of new Safl or ßg / II restriction sites in any target genes. At these restriction sites, for example, an oligonucleotide can be used which codes for an immunologically detectable epitope (28) ("epitope mapping"). The artificially introduced Sall and ßg / II sites in the VBS region (Fig. 2B) are not the only ones possible. Because of the low recognition specificity of cointegrase, further new restriction sites in the "inverted repeats" are possible.
Die folgenden Beispiele erläutern die Erfindung weiter:The following examples further illustrate the invention:
Beispiel 1:Example 1:
12-bp-Linker-Insertionen im arcB-Gen12 bp linker insertions in the arcB gene
Mit dem αrc/i-Gen auf Plasmid pME3659 als Ziel-DNA wurden die in Figur 3A, B gezeigten 12-bp-Insertionen erhalten. Das "Suicide"-Plasmid war pME5 oder pME6. Die Position jeder Insertion wurde durch Nukleotidsequenz- Analyse bestimmt. Ein Vergleich der erhaltenen Insertionsstellen ergab keinerlei Homologie; es gibt keine Anzeichen dafür, daß das IS27-Transpositionssystem eine bestimmte Zielsequenz bevorzugt (12). Unter den Inser- tionsmutanten behielten 45 % eine enzymatische Aktivität; 55 % hatten die Aktivität verloren.With the αrc / i gene on plasmid pME3659 as the target DNA, the 12 bp insertions shown in FIGS. 3A, B were obtained. The "suicide" plasmid was pME5 or pME6. The position of each insertion was determined by nucleotide sequence analysis. A comparison of the insertion sites obtained showed no homology; there is no evidence that the IS27 transposition system prefers a particular target sequence (12). 45% of the insertion mutants retained enzymatic activity; 55% had lost activity.
Unter den aktiven Enzymen hatten zwei neuartige Eigenschaften. Die Insertion bei Position 509 (Fig. 3C) resultierte in einer katabolischen Ornithin-Carbamoyltransferase, welche durch den allosterischen Aktivator AMP (26) nicht mehr aktiviert werden konnte. Die Insertion bei Position 990 (Fig. 3C) ergab ein Enzym, das für sein Substrat Carbamoylphosphat hyperco- operativ war (Hill-Koeffizient nH = 9. statt nH = 3 für das Wildtyp-Enzym, bei pH 6.8).Among the active enzymes, there were two novel properties. The insertion at position 509 (FIG. 3C) resulted in a catabolic ornithine carbamoyl transferase which could no longer be activated by the allosteric activator AMP (26). The insertion at position 990 (FIG. 3C) resulted in an enzyme that was hyperco-operative for its substrate carbamoyl phosphate (Hill coefficient n H = 9 instead of n H = 3 for the wild-type enzyme, at pH 6.8).
Beispiel 2: Inkubation von Ziel-DNAExample 2: Incubation of target DNA
Der Rohextrakt von E. coli ED8767/pME3913 wird nach der Methode von Schmid hergestellt (13). Das Inkubationsgemisch wird auf Eis aus folgenden Komponenten zusammengesetzt:
pME5 (oder pME6) DNA 0.25 μg 2 μlThe crude extract of E. coli ED8767 / pME3913 is produced according to the Schmid method (13). The incubation mixture is composed of the following components on ice: pME5 (or pME6) DNA 0.25 μg 2 μl
Ziel-Plasmid-DNA (z.B. pME3659) 0.25 μg 2 μlTarget plasmid DNA (e.g. pME3659) 0.25 μg 2 μl
Mix 14 μlMix 14 μl
25 mM Hepes pH 7.5 lO mM M9C1225mM Hepes pH 7.5 10mM M9C12
I mM DithiothreitolI mM dithiothreitol
50 mM KCI50 mM KCI
1.8 mM ATP1.8 mM ATP
50μg/ml Bovines Serum-Albumin50μg / ml bovine serum albumin
5 % Polyvinylpyrrolidon K905% polyvinyl pyrrolidone K90
Rohextrakt (40 bis 80 μg Protein) 2 μlCrude extract (40 to 80 μg protein) 2 μl
Totalvolumen 20 μlTotal volume 20 μl
Das Reaktionsgemisch wird 60 Minuten bei 30 °C inkubiert. Danach beträgt das Verhältnis von Cointegraten zu Zielplasmid ca. IO"3. Totale Plasmid-DNA (24) wird je einmal mit Phe¬ nol/ Chloroform (23) und anschließend mit dem Geneclean II-Kit (BIO 101) gereinigt. Ge- reinigte Plasmid-DNA wird durch Elektroporation (27) in E. coli ED8767 eingeführt, mit Se¬ lektion für die Kanamycin-Resistenz von pME5 (bzw. die Chloramphenicol-Resistenz von pME6).The reaction mixture is incubated at 30 ° C for 60 minutes. Thereafter, the ratio of cointegrates to target plasmid is approximately IO "3. Total plasmid DNA (24) is cleaned once with phenol / chloroform (23) and then with the Geneclean II kit (BIO 101). Cleaned Plasmid DNA is introduced into E. coli ED8767 by electroporation (27), with selection for the kanamycin resistance of pME5 (or the chloramphenicol resistance of pME6).
Legende zu den FigurenLegend to the figures
Fig. 1 : In-vivo-Verfahren; Selektion, Reinigung und Auflösung, von Cointegraten. Die wichtigsten Restriktionsschnittstellen sind angegeben. Die Cointegration findet nach der ersten Transformation statt. In den Zellen A ist neben dem Cointegrat noch das Zielplasmid vorhanden. Nach der zweiten Transformation mit verdünnter Plasmid-
DNA enthalten die Zellen B nur noch ein Cointegrat. SS Suicide-Plasmid, z.B. pME6; l=] Zielplasmid, z. B. pME3659. CMR, Chloramphenicol-Resistenz. Schritt nach A: Transformation von ECOLIST mit pME6, Selektion auf Cm50-Me- dium, Cointegrat-BildungFig. 1: In vivo method; Selection, cleaning and dissolution of coin integrates. The most important restriction interfaces are given. The cointegration takes place after the first transformation. In addition to the cointegrate, the target plasmid is also present in cells A. After the second transformation with diluted plasmid DNA B contains only one cointegrate. SS suicide plasmid, eg pME6; l =] target plasmid, e.g. B. pME3659. CM R , chloramphenicol resistance. Step after A: transformation of ECOLIST with pME6, selection for Cm 50 medium, cointegrate formation
Schritt nach B: Isolation von Plasmid-DNA, Verdünnung der DNA IO"2 bis IO"3 und Transformation, Selektion auf Cm50-MediumStep after B: isolation of plasmid DNA, dilution of the DNA IO "2 to IO " 3 and transformation, selection on Cm 50 medium
Schritt nach C: Isolation von Plamsid-DNA, Restriktion mit Safl oder BgRl, Liga¬ tion, TransformationStep after C: isolation of plamsid DNA, restriction with Safl or BgRl, ligation, transformation
Fig. 2: VBS-Plasmide pME5 und pME6. A. Restriktionskarten. Das 0.14 kb große Frag¬ ment, bezeichnet mit VBS-Region. enthält die reaktive IS27-lS27-Verbindung. CMR, Chloramphenicol-Resistenz, ApR, Ampicillin-Resistenz. B. Nukleotidsequenz der VBS-Region. Sie befindet sich auf dem gezeigten 0.14 kb Hmöttll-EcoRI-Fragment. Die gegenüber der IS27 -Wildtypsequenz mutierten Nukleotide sind mit * markiert. Nukleotide, die nicht zur IS27-Sequenz gehören, sind mit ° gekennzeichnet. IR: iriverted repeats.Fig. 2: VBS plasmids pME5 and pME6. A. Restriction maps. The 0.14 kb fragment, designated VBS region. contains the reactive IS27-IS27 connection. CM R , chloramphenicol resistance, Ap R , ampicillin resistance. B. VBS region nucleotide sequence. It is located on the 0.14 kb Hmöttll-EcoRI fragment shown. The nucleotides mutated compared to the IS27 wild-type sequence are marked with *. Nucleotides that do not belong to the IS27 sequence are marked with °. IR: iriverted repeats.
Fig. 3: A, Lokalisation der Insertionen von pME5 und pME6 in arcB-Gen. Die Positionen beziehen sich auf das erste Nukleotid des ctrC-5-Fragmentes (22). Die Pfeile geben die Orientierung an, welche in B definiert ist. ~?~ Anfang der Translation von arcB; Θ Ende der Translation von arcB. B, Definition der Orientierung, nach der Integration von pME5 und pME6 in arcB (1---2 ). C, Sequenzen der Insertionen bei Position 509 und 990. Insertion 509 wurde mit pME6 gemacht und Insertion 990 mit pME5. Die Orientierung der Insertionen ist in B definiert. Die Ziel-Duplikationen sind un¬ terstrichen.3: A, localization of the insertions of pME5 and pME6 in arcB gene. The positions refer to the first nucleotide of the ctrC-5 fragment (22). The arrows indicate the orientation, which is defined in B. ~ ? ~ Start of arcB translation; Θ End of arcB translation. B, definition of orientation, after the integration of pME5 and pME6 in arcB ( 1 --- 2 ). C, sequences of insertions at position 509 and 990. Insertion 509 was made with pME6 and insertion 990 with pME5. The orientation of the insertions is defined in B. The target duplications are underlined.
Fig. 4: In-vitro-Verfahren, als Suicide-Plasmid ist pME5 gezeigt. A, Die für die Reaktion benötigten IstA(P45)- und IstB-Proteine werden mit Hilfe eines tαc-Promoters, der vor die IS27-Gene kloniert wurde, überproduziert. Ap , Ampicillin-Resistenz. B,
pME3913 wird für die Überproduktion der IS27-Proteine verwendet. Die von der IS27 -Wildtypsequenz abweichende Sequenz wird unten angegeben. In pME3913 sind die RBS 1 und das Startcodon von IstA(P46) deletiert (13). RBS, Ribosomen- bindungsstelle *, Position auf der IS27-Sequenz (12).Fig. 4: In vitro method, pME5 is shown as a suicide plasmid. A, The IstA (P45) and IstB proteins required for the reaction are overproduced using a tαc promoter which has been cloned in front of the IS27 genes. Ap, ampicillin resistance. B, pME3913 is used for the overproduction of the IS27 proteins. The sequence deviating from the IS27 wild type sequence is given below. In pME3913 the RBS 1 and the start codon of IstA (P46) have been deleted (13). RBS, ribosome binding site *, position on the IS27 sequence (12).
Literaturliterature
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Claims
1. Plasmide pME5 und pME6 mit reaktiver Verbindungssequenz gemäß Karte und Nukleo¬ tidsequenz von Fig. 2.1. Plasmids pME5 and pME6 with a reactive connection sequence according to the map and nucleotide sequence from FIG. 2.
2. E. coli Wirtsstamm, dadurch gekennzeichnet, daß die istA(P45)- und istB-Gene von IS27 im Chromosom integriert und induzierbar sind.2. E. coli host strain, characterized in that the istA (P45) and istB genes from IS27 are integrated and inducible in the chromosome.
3. Verfahren zur Herstellung von Linker-Insertionen mittels IS27-Funktionen in vivo, beru¬ hend auf (a) einem Plasmid gemäß Anspruch 1 und (b) einem E. coli Wirtsstamm, welcher dadurch gekennzeichnet ist, daß die istA(P45)- und istB-Gene von IS27 im3. A method for producing linker insertions by means of IS27 functions in vivo, based on (a) a plasmid according to claim 1 and (b) an E. coli host strain, which is characterized in that the istA (P45) - and is B genes from IS27 im
Chromosom integriert und induzierbar sind.Chromosome are integrated and inducible.
4. Verfahren zur Herstellung, von Linker-Insertionen mittels IS27-Funktionen in vitro, be¬ ruhend auf (a) einem Plasmid gemäß Anspruch 1 und (b) einem Rohextrakt, enthaltend die überproduzierten Proteine IstA(P45) und IstB von IS27. 4. Process for the production of linker inserts by means of IS27 functions in vitro, based on (a) a plasmid according to claim 1 and (b) a crude extract containing the overproduced proteins IstA (P45) and IstB from IS27.
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PCT/EP1996/003625 WO1997007220A2 (en) | 1995-08-18 | 1996-08-16 | Linker insertion mutagenesis by means of the transposition system of insertion sequence is21 |
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WO (1) | WO1997007220A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6686713B2 (en) | 2001-09-26 | 2004-02-03 | S.N.R. Roulements | Pulse-controlled electric power-assisted steering system |
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1996
- 1996-08-16 WO PCT/EP1996/003625 patent/WO1997007220A2/en active Application Filing
Non-Patent Citations (3)
Title |
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BACTERIAL CONJUGATION (CLEWELL, D., ed.), PLENUM PRESS, New York, 1993; Chapter 6; REIMMANN, C. & HAAS, D.:'Mobilization of chromosomes and nonconjugative plasmids by cointegrative mechanisms.' XP000616906 in der Anmeldung erw{hnt * |
Diss. ETH Nr. 10197. Z]RICH 1993. SCHMID, S.:'Die Insertionssequenz IS21: Entwicklung eines In-vitro Integrationssystems' XP002024154 in der Anmeldung erw{hnt * |
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, Bd. 88, Juni 1991, WASHINGTON US, Seiten 5457-5461, XP002024153 HOEKSTRA, M. ET AL.: "A Tn3 derivative that can be used to make short in-frame insertions within genes" in der Anmeldung erw{hnt * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6686713B2 (en) | 2001-09-26 | 2004-02-03 | S.N.R. Roulements | Pulse-controlled electric power-assisted steering system |
Also Published As
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WO1997007220A3 (en) | 1997-03-20 |
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