CA1216531A - Cloning vectors for cyanobacterium - Google Patents
Cloning vectors for cyanobacteriumInfo
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- CA1216531A CA1216531A CA000423616A CA423616A CA1216531A CA 1216531 A CA1216531 A CA 1216531A CA 000423616 A CA000423616 A CA 000423616A CA 423616 A CA423616 A CA 423616A CA 1216531 A CA1216531 A CA 1216531A
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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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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Abstract
ABSTRACT OF THE DISCLOSURE
Shuttle cloning vectors, for the cloning of DNA in both E. coli bacteria and cyanobacteria (blue-green algae) are constructed using a bacterial plasmid with a polylinker containing many different restriction enzyme sites. These shuttle vectors contain an antibiotic resistance gene, an origin of replication for E. coli, an origin of replication for cyanobacteria and a polylinker with many restriction enzyme sites for the insertion of DNA fragments. Plasmid vectors generated by this procedure can be used for genetic engineering of cyanobacteria and to clone and manipulate plant genes, for the genetic engineering of plants.
Shuttle cloning vectors, for the cloning of DNA in both E. coli bacteria and cyanobacteria (blue-green algae) are constructed using a bacterial plasmid with a polylinker containing many different restriction enzyme sites. These shuttle vectors contain an antibiotic resistance gene, an origin of replication for E. coli, an origin of replication for cyanobacteria and a polylinker with many restriction enzyme sites for the insertion of DNA fragments. Plasmid vectors generated by this procedure can be used for genetic engineering of cyanobacteria and to clone and manipulate plant genes, for the genetic engineering of plants.
Description
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This invention relates to genetlc engineering, and more speciically to novel cloning vectors for cloning in both coliforms, e.g. Escherichia coli bacteria and cyanobacteria (blue-green algae).
Plasmids are chromosomaL materials consisting e~sentially of circular DNA chains and carrying genetic information. ~o~ification of the plasmid of a cell, e.g. ~y replacement of a portion of its DNA sequence, i.e. a gene, with a different DN~ sequence or gene, is a normal technique of genetic engineering, to vary the properties, secretions etc. of the host cell. One of the most thoroughly stu~ied cells in terms of geneticst and one wbich reproduces itself very rapidly under tbe appropriate conditions so as rapidly to proàuce quantities of cell contents such as plasmids, is the bacterium E. coli.
Cyanobacteria, also known as blue-green algae, survive and multiply by photosynthesis. In this and other regards, they resemble higher plants. Ho~ever, they gxow and reproduce very much more rapidly than plants, so that st~dies of photosynthesis and the like are more conveniently conducted with blue-green algae. In many respects, blue-green algae mimic the hehavlour of plants so that successful experimental and research results in blue-~reen algae can often be predicted to be applicable to plants. ~n example of this i5 in herbicide resistance. It would be most beneficial to be able to ~uild a degree o~
herbicide (e.g. triazine) resistance into selected plants, genetically. The triaæine sensitive gene, however, can best be
This invention relates to genetlc engineering, and more speciically to novel cloning vectors for cloning in both coliforms, e.g. Escherichia coli bacteria and cyanobacteria (blue-green algae).
Plasmids are chromosomaL materials consisting e~sentially of circular DNA chains and carrying genetic information. ~o~ification of the plasmid of a cell, e.g. ~y replacement of a portion of its DNA sequence, i.e. a gene, with a different DN~ sequence or gene, is a normal technique of genetic engineering, to vary the properties, secretions etc. of the host cell. One of the most thoroughly stu~ied cells in terms of geneticst and one wbich reproduces itself very rapidly under tbe appropriate conditions so as rapidly to proàuce quantities of cell contents such as plasmids, is the bacterium E. coli.
Cyanobacteria, also known as blue-green algae, survive and multiply by photosynthesis. In this and other regards, they resemble higher plants. Ho~ever, they gxow and reproduce very much more rapidly than plants, so that st~dies of photosynthesis and the like are more conveniently conducted with blue-green algae. In many respects, blue-green algae mimic the hehavlour of plants so that successful experimental and research results in blue-~reen algae can often be predicted to be applicable to plants. ~n example of this i5 in herbicide resistance. It would be most beneficial to be able to ~uild a degree o~
herbicide (e.g. triazine) resistance into selected plants, genetically. The triaæine sensitive gene, however, can best be
- 2 - ~i~
identified and hence modifie~ by studies and experimentation with blue-green algae. Then a triazine-resistant gene can be made and cloned in~o the plant. Ordinary bacteria cannot be used for such work, because they do not behave analogously to plan~s.
Moveover, blue-green algae have beneficial utility in their own right. They can fix nitrogen, the natural process by which atmospheric nitrogen is fixed in agricultural soil as ammonia and its derivatives. Efficient natural nitrogen fixation reduces or even eliminates the requirement for chemical fertilizers. At present, bacteria which are symbiotic with legumes, some trees and certain ferns are used to enhance nitrogen fixation. When these are associated with crops, the nitrogen fixation occurs at the expense of reduced crop yieldsO
The agricultural use of blue-green algae for this purpose, with enhanced nitrogen fixation capability, would be beneficial.
Blue-green algae also have potential of providing significant amounts of protein, for focd purposes, using solar energy as the only energy source, on account of their photosynthetic growth. Other edible products such as food dyes are also ob~ainable from blue-green algae.
The present invention provides cloning vectors which will grow in both E. Coli and blue-green algae. The vectors are DNA plasmids. Accordingly, they can be produced comparatively rapidly by cultivation and growth of E. Coli, extracted therefrom and introduced into blue-green algae, to modify the genetic properties thereof. l'hese vectors are small enough that
identified and hence modifie~ by studies and experimentation with blue-green algae. Then a triazine-resistant gene can be made and cloned in~o the plant. Ordinary bacteria cannot be used for such work, because they do not behave analogously to plan~s.
Moveover, blue-green algae have beneficial utility in their own right. They can fix nitrogen, the natural process by which atmospheric nitrogen is fixed in agricultural soil as ammonia and its derivatives. Efficient natural nitrogen fixation reduces or even eliminates the requirement for chemical fertilizers. At present, bacteria which are symbiotic with legumes, some trees and certain ferns are used to enhance nitrogen fixation. When these are associated with crops, the nitrogen fixation occurs at the expense of reduced crop yieldsO
The agricultural use of blue-green algae for this purpose, with enhanced nitrogen fixation capability, would be beneficial.
Blue-green algae also have potential of providing significant amounts of protein, for focd purposes, using solar energy as the only energy source, on account of their photosynthetic growth. Other edible products such as food dyes are also ob~ainable from blue-green algae.
The present invention provides cloning vectors which will grow in both E. Coli and blue-green algae. The vectors are DNA plasmids. Accordingly, they can be produced comparatively rapidly by cultivation and growth of E. Coli, extracted therefrom and introduced into blue-green algae, to modify the genetic properties thereof. l'hese vectors are small enough that
- 3 -r ~..1~
3~
they are easily introd~lce~ into a cell. 'I~hey have a large number of unique restriction enz~me sites which are not part o~
any essential gene. This inventivn al50 provides novel plasmios resulting from ~he com~ination oE ~uch a vector with a plasmid derived from blue-green algae, and a method for nlaki~g them.
According to one aspect of the present invention, a process is provided wherein a ~acterial originating plasmid containing its origin or replication gene, an antibiotic resistance gene or other appropriate markers, and a polylinker having at least five unique restriction endonuclease recognition sites, i5 combined by recombinant techni~ues witb a plasmid from a blue-green algae or cyanobacterium, to produce a new plasmid having the bac~eriaL origin, the cyanobacterium origin and the antibiotic resistance gene, as well ~s a plurality of restriction endonuclease recognition sites on the polylinker.
Typically and preferably, the bacterial originating plasmid is derived from E. coli.
In another aspect of the present invention, the new plasmid is subse~uently reduced in size, by use of appropriate endonucleases followed by ligases to remove tnerefrom inessential DNA sequences whilst maintaining intact the bacterial origin and the cyanobacterial origin and the antibiotic resistance gene. In such a manner, a plasmid of suitable size for ready introduction into viable plant or blue-green algae cells can be made.
Polylinkers in general terms have been created previously for use in genetic engineering and molecular cloning. They are segments o~ DNA that contain closely spacea sites for many different restriction enzymes.
Pol~linker-containing plasmids for the present invention can be prepared from commercially available, known plasmids, ~y use of restriction enzyme techni~ues to cut -tne DWA
chain of the commercial plasmid at the required location, and b~
inserting into the cut ~NA chain a preformed natural or artificial polylinker sequence using DN~ ligase in tne usual way. The result is an h. coli plasmid containing an artificially produced polylinker. For maximum versatility in subsequent use and applications, it is preferred tnat the polylinker used in the present invention sbould have as large a number of endonuclease sites as possible, without introducing superfluous DNA sequences.
In order that two plasmids or other DNA chains may be cut and the resulting fragments recombined together to form ne~
recômbinant DN~ chain sequences therefrom, it lS necessary that both orginating plasmids be provided with restriction enzyme recognition si~es in the chain which, after enzynlatic cleavage, leave mutuall~ compatible chain ends for ligation. Pre~era~ly both originating plasmids are provided with the same restriction enzyme recognition site. Then, both plasmids can be cleaved b~
use of the same restriction enz~me~ and the fragments so formed will have mutually compatible end groupings ("sticky ends") as a result of cleavage by the same enzyme, and can recombine with one another. The inserted segment can also be recovered from such an arrangement. To ren~er a plasmid recombina~le with ~g~33.~
greatest variety of other plasmids, i.e. to increase its versatility, it should be provided with the greatest diversity of restriction enzyme sensitive sites. Normal~y, this means providing a plasmid of large size (large numbers of base pairs) to accomodate a sufficiently large number of such sites to provide the desired versatility, but by use of polylinkers according to the present invention the size of the plasmid can be substantially reduced without sacrifice of versatility.
In the accompanying drawings:
Figure 1 is a diagrammatic illustration of a polylinker containing plasmid example according to the present invention;
Figure 2 is a diagrammatic illustration of a natural plasmid from a bl~e-green algae, useful Eor combining with the vector shown in FigO l;
Figure 3 is a diagrammatic illustration of a plasmi~
cloning vector resulting from interaction of the plasmids of Figure 1 and Figure 2;
Figure 4 is a ~imilar diagrammatic illustra~ion of an alternative embodiment of a cloning vector prepared from that shown in Figure 3;
Figure 5 is another cloning vector according to the present invention, prepared by interaction of the vector of Figure 4 witn that of ~ig. 1;
~ igure ~ is a diagrammatic process flow sheet of processes according to the present invention.
A spec1fic example of a commercially available plasmld use~ul as a starting material in the present invention is tha-t known as pBR 322, an E.coli originating plasmid which is well known and has been f~lly sequenced.
Cloning vector PBR 322 is perhaps the most widely used E coli vector. It is a plasmid under relaxed control o~ DNA
synthesis that contains both ampicillin- and tetracycline-resistance yenes and a number o~ convenient restriction sites ItS complete nucleotide se~uence and genetic map are known, and published by J.G. Sutcli~fe~ Cold ~pring Harbour Symposium 43, p.77 (1979).
According to a specific example of the present invention, PBR 322 has been modified b~ replacement of the tetracycline resistance qene with a polylinker ha~ing 7g base pairs and 13 restriction endonuclease cloning sites, and the deletion o~ ot~er non-essential DNA. The modified product~ a cloning vector, is illustrated diagrammatically in Figure 1.
The inner circle (10) thereof represents the scale of the plasmid chain, in thousand base pairs, and is not part of the chemical structure. The larger, outer circle (12) represents the residual DNA chain of the pB~ 322 plasmid, with some of the residual unique restriction endonuclease sites thereon. The portion of the cb~in (12) designatea AMP represents the gene of ampicillin-resistance thereon. The upper, arcuate portion (14) represents the polylinker, which constitutes part of the plasmid chain (12) and bears a large number of restriction endonuclease sites as shown. The symbols used on Elig. 1 for res~riction endonuclease recognition sites are the standard~
well-known desiqnations for the appropriate restric~lon en2ymes. The polylinker (l4) has been spliced into the main DNA
chain of plasmid pBR 322 by normal recombinant techni~ues, using restriction enzymes to cleave the plasmid DNA chain, addition of the polylinker and use of DNA ligase to recombine the polylinker sequence and the plasmid DNA chain into a single plasmid referred to herein as pDPL 13.
This plasmid pDPL 13 lS a versatile cloning vector containing a polylinker with 13 endonuclease cloning sites along a chain of ~9 base pairs. It can be used to construct a variety of other plasmids that act as shuttle vectors witn oriyins o~
replication for both E coli and various blue green algae, for example Anacystis nidulans. The shuttle vectors so formed can be used to introduce potentially commercially important plant genes in cyanobacteria where genes can function. They can also be used to engineer new blue-green algae.
Preferably~ the shuttle vectors are prepared by combining plasmid p~PL 13 with a naturally occurring plasmid from the cyanobacterium. Many species of cyanobacterium have plasmids which can be combined with other plasmids in a similar manner. A speci~ic example of a suitable such plasmid is the smaller of the two plasmids naturally occurring in and extractible from the cyano~acterium species Anacystis nidulans, and referred to herein as pANS. T~iS plasmid is diagrammatically illustrated in Fig. 2, in the same general format as Fig. l. The DNA chain of pANS and the polylin~er o~
pDPL 13 both contain a Bam Hl endonuclease restriction sites as 3~
sno~n in the drawings, which are cut by addition to a mlxture o~
the plasmids, under appropriate conditions, of Bam endonuclease. Then an appropriately controlle~ reac~lon of the fragment-containing mixture with DNA ligase causes recombination thereof, to form the synthetic plasmid of Eig. 3, herein designated, pPLANB2. It will be noted that the polylinker (14) of pDPL 13 has been split into a 25 base pair sequence and a 54 base pair sequence, which are separated on ~he pPLAN~2 plasmid by the ampicillin resistance gene.
Whilst for speci$ic illustrative purposes, Bam Hl endonuclease was chosen and used ~or cutting purposes, it is within the scope of the pre~err@d embodiment of ~his inventiQn to use any other restriction enzyme capable of cutting both the DNA chain of tne cyanobacteria-originating plasmid and the DNA
chain of the pDPL 13 synthetic plasmid, so as to prod~ce recombinable frac~ions from each.
Tne resulting product i5 effectively a combination of all or part of a cyanobacterium plasmid and a plasmid originating fronl a bacterium with a polylinker se~uence, of sufficiently small size easily to enter a cell, e.g. of a plant, cyanobacterium or bacteria, yet capable of being maintained in either cyanobacteria or bacteria. The advantages of the rapid, ready production of suitable plasmids by use of the E. Coli bacterium, in generating pDPL 13, have been retained.
The cloning vector plasmid pPLANB~ so fornled, as illustrated in Fig. 3, can then be further trimmed to produce alternative cloning vectors. 'rhere are, tor example, a plurality of Xno 1 sltes ~n p~LANB2, both on the polylink~r segment and on the main DNA c~ain. '~hus, r~action thereof with endonuc]ease Xho 1 will cause DNA chain cleavage at such sites, and then addition to the fragsnent containing mixture of a DN~-ligase, in the standara way, will cause recombination of the fr3gments. As a result, a cloning vector plasmid as shown in Fig. 4 can be ~ormed, herein referred to as pCB4. This plasmid retains a substantial number of unique restriction enzyme (cloning) sites, allowing for versatility thereof, and retains its ampicillin resistant gene. It i5 however substantiall~
smaller than pPLANB2, having about ~./ k base pairs as opposed to about 10.2 k base pairs, so that it is more readily insertable into viable cells, and can accept a larger fragment of exogenous DNA.
As an example of its versatility, plasmi~ pCB4 may be further modified/ by treatment with restriction nucleases towards which it has two or more sensitive sites, with recombination with the original pol~linker-plasmid pDPL13 to produce new clvnin~ vector plasmids of great versatility. ThUs, reaction pCB4 with restriction enzymes Clal, will cause pCB4 to cleave at two places, to form two different plasmid DNA
fragments, which can be isolated fronl one another by known techniques. Plasmid pDPL13 as shown in Fig. 1, can also be cleaved with Clal. The appropriate isolated fragment of CLAl cleaved pCB4 may then be mixed with CLAl-cleaved plasmid pDPIJ13, and reacted with DNA ligase, to form a new cloning vector plasmid as illustrated in Eig. 5. This and the sequence of various other process ~teps described above is diagrammaticall~ illustrated in Fi9. 6. r~his plasmid, re~erred to herein as~6k~bb contains a polylinker having 100 ~ase pairs with 17 restriction enzyme or cloning sites, in a plasmid of overall si~e only about 5 K base pairs. The plasnlia o~ this siæe is readily introduced into E. coli cells, ana has been found to replicate therein, so ~hat large ~uanti~ies of it can be produced in this manner. It retains the original replication origin of the original pANS natural plasmid from the blue-green algae (cyanobacterium) Ana~ySti9 nidulans, and can be used directly therein to enhance characteristic functions thereof.
At the same time, it retains a substantial number ancl variety of restriction enzyme sensitive or cloning sites, so that it is vers~tile, and can be joined with a substantial variety of DN~
fragments that bave been cut ~ith restriction enzymes so as to produce ligatable endings.
Laboratory tec~ni~ues used for DNA trimming, cnain cleavage, ligationt recombination etc. are generally in accordance with those normally followed by a person skilleo in the art. Many of the techniques employed are described in "~olecular Cloning, a Laboratory Manual!' by T. Maniatis et al, Cold Spring Harbor Laboratory, I982~
3~
they are easily introd~lce~ into a cell. 'I~hey have a large number of unique restriction enz~me sites which are not part o~
any essential gene. This inventivn al50 provides novel plasmios resulting from ~he com~ination oE ~uch a vector with a plasmid derived from blue-green algae, and a method for nlaki~g them.
According to one aspect of the present invention, a process is provided wherein a ~acterial originating plasmid containing its origin or replication gene, an antibiotic resistance gene or other appropriate markers, and a polylinker having at least five unique restriction endonuclease recognition sites, i5 combined by recombinant techni~ues witb a plasmid from a blue-green algae or cyanobacterium, to produce a new plasmid having the bac~eriaL origin, the cyanobacterium origin and the antibiotic resistance gene, as well ~s a plurality of restriction endonuclease recognition sites on the polylinker.
Typically and preferably, the bacterial originating plasmid is derived from E. coli.
In another aspect of the present invention, the new plasmid is subse~uently reduced in size, by use of appropriate endonucleases followed by ligases to remove tnerefrom inessential DNA sequences whilst maintaining intact the bacterial origin and the cyanobacterial origin and the antibiotic resistance gene. In such a manner, a plasmid of suitable size for ready introduction into viable plant or blue-green algae cells can be made.
Polylinkers in general terms have been created previously for use in genetic engineering and molecular cloning. They are segments o~ DNA that contain closely spacea sites for many different restriction enzymes.
Pol~linker-containing plasmids for the present invention can be prepared from commercially available, known plasmids, ~y use of restriction enzyme techni~ues to cut -tne DWA
chain of the commercial plasmid at the required location, and b~
inserting into the cut ~NA chain a preformed natural or artificial polylinker sequence using DN~ ligase in tne usual way. The result is an h. coli plasmid containing an artificially produced polylinker. For maximum versatility in subsequent use and applications, it is preferred tnat the polylinker used in the present invention sbould have as large a number of endonuclease sites as possible, without introducing superfluous DNA sequences.
In order that two plasmids or other DNA chains may be cut and the resulting fragments recombined together to form ne~
recômbinant DN~ chain sequences therefrom, it lS necessary that both orginating plasmids be provided with restriction enzyme recognition si~es in the chain which, after enzynlatic cleavage, leave mutuall~ compatible chain ends for ligation. Pre~era~ly both originating plasmids are provided with the same restriction enzyme recognition site. Then, both plasmids can be cleaved b~
use of the same restriction enz~me~ and the fragments so formed will have mutually compatible end groupings ("sticky ends") as a result of cleavage by the same enzyme, and can recombine with one another. The inserted segment can also be recovered from such an arrangement. To ren~er a plasmid recombina~le with ~g~33.~
greatest variety of other plasmids, i.e. to increase its versatility, it should be provided with the greatest diversity of restriction enzyme sensitive sites. Normal~y, this means providing a plasmid of large size (large numbers of base pairs) to accomodate a sufficiently large number of such sites to provide the desired versatility, but by use of polylinkers according to the present invention the size of the plasmid can be substantially reduced without sacrifice of versatility.
In the accompanying drawings:
Figure 1 is a diagrammatic illustration of a polylinker containing plasmid example according to the present invention;
Figure 2 is a diagrammatic illustration of a natural plasmid from a bl~e-green algae, useful Eor combining with the vector shown in FigO l;
Figure 3 is a diagrammatic illustration of a plasmi~
cloning vector resulting from interaction of the plasmids of Figure 1 and Figure 2;
Figure 4 is a ~imilar diagrammatic illustra~ion of an alternative embodiment of a cloning vector prepared from that shown in Figure 3;
Figure 5 is another cloning vector according to the present invention, prepared by interaction of the vector of Figure 4 witn that of ~ig. 1;
~ igure ~ is a diagrammatic process flow sheet of processes according to the present invention.
A spec1fic example of a commercially available plasmld use~ul as a starting material in the present invention is tha-t known as pBR 322, an E.coli originating plasmid which is well known and has been f~lly sequenced.
Cloning vector PBR 322 is perhaps the most widely used E coli vector. It is a plasmid under relaxed control o~ DNA
synthesis that contains both ampicillin- and tetracycline-resistance yenes and a number o~ convenient restriction sites ItS complete nucleotide se~uence and genetic map are known, and published by J.G. Sutcli~fe~ Cold ~pring Harbour Symposium 43, p.77 (1979).
According to a specific example of the present invention, PBR 322 has been modified b~ replacement of the tetracycline resistance qene with a polylinker ha~ing 7g base pairs and 13 restriction endonuclease cloning sites, and the deletion o~ ot~er non-essential DNA. The modified product~ a cloning vector, is illustrated diagrammatically in Figure 1.
The inner circle (10) thereof represents the scale of the plasmid chain, in thousand base pairs, and is not part of the chemical structure. The larger, outer circle (12) represents the residual DNA chain of the pB~ 322 plasmid, with some of the residual unique restriction endonuclease sites thereon. The portion of the cb~in (12) designatea AMP represents the gene of ampicillin-resistance thereon. The upper, arcuate portion (14) represents the polylinker, which constitutes part of the plasmid chain (12) and bears a large number of restriction endonuclease sites as shown. The symbols used on Elig. 1 for res~riction endonuclease recognition sites are the standard~
well-known desiqnations for the appropriate restric~lon en2ymes. The polylinker (l4) has been spliced into the main DNA
chain of plasmid pBR 322 by normal recombinant techni~ues, using restriction enzymes to cleave the plasmid DNA chain, addition of the polylinker and use of DNA ligase to recombine the polylinker sequence and the plasmid DNA chain into a single plasmid referred to herein as pDPL 13.
This plasmid pDPL 13 lS a versatile cloning vector containing a polylinker with 13 endonuclease cloning sites along a chain of ~9 base pairs. It can be used to construct a variety of other plasmids that act as shuttle vectors witn oriyins o~
replication for both E coli and various blue green algae, for example Anacystis nidulans. The shuttle vectors so formed can be used to introduce potentially commercially important plant genes in cyanobacteria where genes can function. They can also be used to engineer new blue-green algae.
Preferably~ the shuttle vectors are prepared by combining plasmid p~PL 13 with a naturally occurring plasmid from the cyanobacterium. Many species of cyanobacterium have plasmids which can be combined with other plasmids in a similar manner. A speci~ic example of a suitable such plasmid is the smaller of the two plasmids naturally occurring in and extractible from the cyano~acterium species Anacystis nidulans, and referred to herein as pANS. T~iS plasmid is diagrammatically illustrated in Fig. 2, in the same general format as Fig. l. The DNA chain of pANS and the polylin~er o~
pDPL 13 both contain a Bam Hl endonuclease restriction sites as 3~
sno~n in the drawings, which are cut by addition to a mlxture o~
the plasmids, under appropriate conditions, of Bam endonuclease. Then an appropriately controlle~ reac~lon of the fragment-containing mixture with DNA ligase causes recombination thereof, to form the synthetic plasmid of Eig. 3, herein designated, pPLANB2. It will be noted that the polylinker (14) of pDPL 13 has been split into a 25 base pair sequence and a 54 base pair sequence, which are separated on ~he pPLAN~2 plasmid by the ampicillin resistance gene.
Whilst for speci$ic illustrative purposes, Bam Hl endonuclease was chosen and used ~or cutting purposes, it is within the scope of the pre~err@d embodiment of ~his inventiQn to use any other restriction enzyme capable of cutting both the DNA chain of tne cyanobacteria-originating plasmid and the DNA
chain of the pDPL 13 synthetic plasmid, so as to prod~ce recombinable frac~ions from each.
Tne resulting product i5 effectively a combination of all or part of a cyanobacterium plasmid and a plasmid originating fronl a bacterium with a polylinker se~uence, of sufficiently small size easily to enter a cell, e.g. of a plant, cyanobacterium or bacteria, yet capable of being maintained in either cyanobacteria or bacteria. The advantages of the rapid, ready production of suitable plasmids by use of the E. Coli bacterium, in generating pDPL 13, have been retained.
The cloning vector plasmid pPLANB~ so fornled, as illustrated in Fig. 3, can then be further trimmed to produce alternative cloning vectors. 'rhere are, tor example, a plurality of Xno 1 sltes ~n p~LANB2, both on the polylink~r segment and on the main DNA c~ain. '~hus, r~action thereof with endonuc]ease Xho 1 will cause DNA chain cleavage at such sites, and then addition to the fragsnent containing mixture of a DN~-ligase, in the standara way, will cause recombination of the fr3gments. As a result, a cloning vector plasmid as shown in Fig. 4 can be ~ormed, herein referred to as pCB4. This plasmid retains a substantial number of unique restriction enzyme (cloning) sites, allowing for versatility thereof, and retains its ampicillin resistant gene. It i5 however substantiall~
smaller than pPLANB2, having about ~./ k base pairs as opposed to about 10.2 k base pairs, so that it is more readily insertable into viable cells, and can accept a larger fragment of exogenous DNA.
As an example of its versatility, plasmi~ pCB4 may be further modified/ by treatment with restriction nucleases towards which it has two or more sensitive sites, with recombination with the original pol~linker-plasmid pDPL13 to produce new clvnin~ vector plasmids of great versatility. ThUs, reaction pCB4 with restriction enzymes Clal, will cause pCB4 to cleave at two places, to form two different plasmid DNA
fragments, which can be isolated fronl one another by known techniques. Plasmid pDPL13 as shown in Fig. 1, can also be cleaved with Clal. The appropriate isolated fragment of CLAl cleaved pCB4 may then be mixed with CLAl-cleaved plasmid pDPIJ13, and reacted with DNA ligase, to form a new cloning vector plasmid as illustrated in Eig. 5. This and the sequence of various other process ~teps described above is diagrammaticall~ illustrated in Fi9. 6. r~his plasmid, re~erred to herein as~6k~bb contains a polylinker having 100 ~ase pairs with 17 restriction enzyme or cloning sites, in a plasmid of overall si~e only about 5 K base pairs. The plasnlia o~ this siæe is readily introduced into E. coli cells, ana has been found to replicate therein, so ~hat large ~uanti~ies of it can be produced in this manner. It retains the original replication origin of the original pANS natural plasmid from the blue-green algae (cyanobacterium) Ana~ySti9 nidulans, and can be used directly therein to enhance characteristic functions thereof.
At the same time, it retains a substantial number ancl variety of restriction enzyme sensitive or cloning sites, so that it is vers~tile, and can be joined with a substantial variety of DN~
fragments that bave been cut ~ith restriction enzymes so as to produce ligatable endings.
Laboratory tec~ni~ues used for DNA trimming, cnain cleavage, ligationt recombination etc. are generally in accordance with those normally followed by a person skilleo in the art. Many of the techniques employed are described in "~olecular Cloning, a Laboratory Manual!' by T. Maniatis et al, Cold Spring Harbor Laboratory, I982~
Claims (9)
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for preparing shuttle cloning vectors, for the cloning of DNA in both E. Coli and cyanobacteria, which comprises combining together a plasmid from a cyanobacterium which contains the replication gene thereof, with a plasmid from E. Coli, said E. Coli plasmid including the replication gene thereof, at least one antibiotic resistance gene, and a polylinker DNA segment having at least five unique restriction enzyme recognition sites thereon, said plasmids being initially cleaved in a manner which leaves intact the replication genes and antibiotic resistance genes thereof, and produces compatible DNA chain ends thereon, and then combined together to produce a recombinant plasmid with both replication genes and the antibiotic resistance genes.
2. The process of claim 1 wherein both plasmids are cleaved with the same endonuclease and the fragments so produced then recombined with DNA ligase.
3. The process of claim 2 wherein the recombinant plasmid so formed is subsequently treated with an appropriate endonuclease for which the recombinant plasmid has two recognition sites, and recombining the cleaved DNA segments so formed which contain the replication genes and the antibiotic resistance genes, so as to obtain a second recombinant plasmid of reduced DNA chain length.
4. The process of claim 3 wherein said second recombinant plasmid is further combined with an E. coli derived bacterial plasmid including the replication gene thereof, an antibacterial resistance gene and a polylinker DNA segment having at least five unique restriction enzyme recognition sites thereon.
5. A shuttle cloning vector comprising a recombinant plasmid derived from recombination of DNA fragments cleaved from a cyanobacterium plasmid and an E. coli derived plasmid, said recombinant plasmid including an E. coli replication gene, a cyanobacterium replicating gene and an antibiotic resistance gene.
6. The vector of claim 5, further including a polylinker segment providing at least five restriction enzyme recognition sites in a fifty base pair DNA chain segment.
7. The shuttle cloning vector pPLAN B2 as diagrammatically illustrated in Fig. 2 of the accompanying drawings.
8. The shuttle cloning vector pCB4 as diagrammatically illustrated in Fig. 4 of the accompanying drawings.
9. The shuttle cloning vector pANSO1 as diagrammatically illustrated in Fig. 5 of the accompanying drawings.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000423616A CA1216531A (en) | 1983-03-15 | 1983-03-15 | Cloning vectors for cyanobacterium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000423616A CA1216531A (en) | 1983-03-15 | 1983-03-15 | Cloning vectors for cyanobacterium |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1216531A true CA1216531A (en) | 1987-01-13 |
Family
ID=4124784
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000423616A Expired CA1216531A (en) | 1983-03-15 | 1983-03-15 | Cloning vectors for cyanobacterium |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1216531A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2637293A1 (en) * | 1988-07-26 | 1990-04-06 | Commissariat Energie Atomique | Recombinant plasmids, vectors comprising the said plasmids, processes for producing them, modified cyanobacterium strain Synechocystis capable of being host of the said plasmids and/or of the said vectors |
| EP0533942A1 (en) * | 1991-03-13 | 1993-03-31 | HAGIWARA, Yoshihide | Process for expressing polypeptide |
| US5804408A (en) * | 1991-03-13 | 1998-09-08 | Yoshihide Hagiwara | Expression of human SOD in blue green algae |
-
1983
- 1983-03-15 CA CA000423616A patent/CA1216531A/en not_active Expired
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2637293A1 (en) * | 1988-07-26 | 1990-04-06 | Commissariat Energie Atomique | Recombinant plasmids, vectors comprising the said plasmids, processes for producing them, modified cyanobacterium strain Synechocystis capable of being host of the said plasmids and/or of the said vectors |
| EP0533942A1 (en) * | 1991-03-13 | 1993-03-31 | HAGIWARA, Yoshihide | Process for expressing polypeptide |
| EP0533942A4 (en) * | 1991-03-13 | 1994-06-01 | Hagiwara Yoshihide | Process for expressing polypeptide |
| US5804408A (en) * | 1991-03-13 | 1998-09-08 | Yoshihide Hagiwara | Expression of human SOD in blue green algae |
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