Note: Descriptions are shown in the official language in which they were submitted.
"Y~
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
- 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~
