Hogg S. Essential microbiology

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318 MICROORGANISMS IN GENETIC ENGINEERING

ector

Donor DNA

RE digestion

Recombinant vector

Host bacterial cell V

ector replication

Bacterial replication

Clone of identical cells

Figure 12.3 Gene cloning. DNA fragments obtained by restriction digestion can be spliced

into a similarly digested vector molecule and transformed into a host bacterial cell. See the

text for details

A selectable marker is

a gene that allows cells

containing it to be identi-

fied by the expression of

a recognisable charac-

teristic.

it must carry a selectable marker – since only a pro-

portion of host cells will take up the vector, there must

be a means of differentiating them from those that do

not. A common way to do this is to use a vector that

carries a gene that confers resistance to an antibiotic

such as ampicillin. When bacterial cells are plated out

on a medium containing the antibiotic, only those that

have taken up the vector will be able to form colonies.

(The host strain must, of course, normally be suscep-

tible to the antibiotic.)

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PLASMID CLONING VECTORS 319

it must carry a single copy of RE restriction sites – in order to accommodate a piece

of donor DNA, a vector must be cut by a restriction endonuclease in one place only

(Figure 12.3).

Plasmid cloning vectors

A multiple cloning site

or polylinker is a region

of a cloning vector de-

signed to contain recog-

nition sequences for sev-

eral REs.

Two main types of vector system are used in cloning,

those that use plasmids and those that use bacterio-

phages (revisit Chapters 3 and 10, respectively, for a

reminder of the main features of these). Naturally occur-

ring examples of these are manipulated so that they pos-

sess the above properties. A popular vector in the early

days of gene cloning was the plasmid pBR322; Figure

12.4 shows how it contains the features described above.

The lacZ



gene actu-

ally only encodes a part

of the β-galactosidase

enzyme, called the α-

peptide. The strain of E.

coli used as host makes

an incomplete version of

the enzyme, which lacks

this portion. Only if the

cells contain the plasmid

with the lac Z’ gene can

they produce functional

β-galactosidase, by α-

complementation.

Let us consider now what happens, at a molecular

level, when donor DNA is ligated into a plasmid vector

(Figure 12.5). Sticky ends of the donor fragment form hy-

drogen bonds with the exposed compatible ends of the

opened up plasmid by complementary base pairing, and

DNA ligase consolidates the join. Many plasmids con-

tain engineered sequences called multiple cloning sites

(MCS); these provide additional ﬂexibility with respect

to the restriction fragments that may be accommodated.

The ligation of insert DNA into the cloning site of

the plasmid is not the only possible outcome of the pro-

cedure described above, however. Unless experimental

conditions are carefully controlled (there are ways of

doing this), a more likely outcome is that the two com-

patible ends of the plasmid will simply ‘ﬁnd’ each other

Figure 12.4 Plasmid pBR322. One of the earliest plasmid vectors, pBR322 illustrates the

major features required for use in gene cloning: an origin of replication (ori), selectable

markers (genes for resistance to ampicillin and tetracycline), and single recognition sites for

a number of REs

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320 MICROORGANISMS IN GENETIC ENGINEERING

Recognition sites

for restriction

endonuclease (RE)

Digestion with

RE fragments

donor DNA and

opens up

vector

Donor

DNA

Formation of

recombinant vector

molecule. DNA

ligase consolidates

the join by repairing

the phosphate-sugar

ackbone.

..…G

..…CCTAG

GATCC…..

G…..

Compatible ‘sticky ends’

Figure 12.5 Formation of a recombinant plasmid. A recombinant plasmid is formed when

a fragment of foreign DNA is taken up and ligated into the plasmid. By cleaving both plasmid

and foreign DNA with the same RE, compatible ‘sticky’ ends are created, facilitating the

join. Treatment with the enzyme alkaline phosphatase prevents the cut plasmid ends rejoining

together, thereby favouring the formation of recombinant molecules

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PLASMID CLONING VECTORS 321

pUC19

Fragment with

BamHI sticky

ends

ori

lacZ’amp

pUC19

BamH1

recognition

site

Inserted

fragment

Transform into E. coli cells

and plate onto medium

containing ampicillin and

Xgal (artificial substrate

for β-galactosidase)

Recombinant colony

(white)

Non-recombinant

colony (blue)

Figure 12.6 Recombinant plasmids can be detected by insertional inactivation. Insertion

of foreign DNA is carried out at a site within one of the selectable markers, thus interrupting

its gene sequence. Here, a fragment has been inserted into the BamHI site situated within

the gene that codes for β-galactosidase. Bacteria transformed with such a plasmid will not

produce the functional enzyme, and so can be distinguished from those carrying plasmids

with no inserted DNA

again, and rejoin. Since a certain amount of this is inevitable, how are we able to

tell the difference between those bacteria that contain a recombinant plasmid (one

containing a piece of donor DNA) and those that have taken up a recircularised ‘native’

plasmid? Since both types will contain the gene for ampicillin resistance, we cannot

distinguish them by this means. A strategy commonly used to get around the problem

is insertional inactivation (Figure 12.6). This clever ploy exploits the fact that we can

manipulate DNA, and, for example, insert RE recognition sequences at desired points.

If a recognition site occurs in the middle of a gene sequence, and a piece of foreign

DNA is inserted at this position, the gene will be interrupted, and unable to produce

a functional gene product. In the example shown, the gene is lacZ



, necessary for the

successful expression of the enzyme β-galactosidase. This will only be expressed in

those bacteria that contain plasmids in which the gene has remained uninterrupted,

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322 MICROORGANISMS IN GENETIC ENGINEERING

i.e. those that have not taken up an insert. Expression of the β-galactosidase can be

detected by growing the bacteria on an artiﬁcial substrate, which is converted to a

coloured (usually blue) product when acted on by the enzyme. Those cells that contain

recombinant plasmids are easily identiﬁed because disruption of the lacZ



gene means

that no β-galactosidase is produced, resulting in non-pigmented (white) colonies.

One problem remains. Remember that our inserted DNA was derived from the di-

gestion of total (genomic) DNA from the donor organism; this means that our DNA

library will contain recombinant plasmids with a whole range of fragments from that

digestion, and not just the speciﬁc one that interests us. How are we able to distinguish

this fragment from the others?

A technique called nucleic acid hybridisation is used to solve the problem. This once

again depends on complementary base pairing, and involves the creation of a probe,

a short length of single-stranded DNA that is complementary to part of the desired

‘target’ sequence, and therefore able to seek it out. If searching for the right clone can

be likened to looking for a needle in a haystack, then the probe is a powerful ‘magnet’

that makes the task much easier. The probe carries a tag or label, so that its location

can be identiﬁed (Figure 12.7).

Once we have identiﬁed the clone of bacteria containing plasmids with the insert that

interests us, we can grow a pure culture of it and then isolate plasmid DNA. Using the

same RE as before, the inserted donor DNA can be removed and puriﬁed. We now have

enough of this speciﬁc DNA sequence (a tiny proportion of the donor organism’s total

genome) to analyse and manipulate.

Nitrocellulose

Petri dish with colonies

of bacteria containing

recombinant plasmids

Peel sheet to

produce replica

of colonies

Lyse bacteria

and denature

DNA

DNA bound

Incubate

with probe

and wash

Expose

to film

a) b)

Figure 12.7 Colony probing. (a) A replica of the bacterial colonies is made using a nitro-

cellulose membrane. (b) Alkali treatment lyses the cells and denatures the DNA, making it

single stranded. (c) Following a period of incubation with a radiolabelled probe to allow

hybridisation to take place, the membrane is washed to remove any unbound radioactivity.

(d) Position of bound radioactivity revealed by autoradiography. Comparison with original

master plate reveals the location of colonies carrying speciﬁc target sequence. Alternative

detection systems such as biotin–streptavidin that avoid the use of radioactivity have be-

come much more commonly used in recent years. From Reece, RJ: Analysis of Genes and

Genomes, John Wiley & Sons, 2003. Reproduced by permission of the publishers

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BACTERIOPHAGES AS CLONING VECTORS 323

Although plasmids are easily isolated and manipulated, their use as cloning vectors

is limited by the fact that they tend to become unstable if we attempt to insert much

more than about 5kb of foreign DNA. For inserts larger than this, we must turn to other

vector systems.

Bacteriophages as cloning vectors

The most commonly used bacteriophage vectors are those based on phage Lambda (λ)

(Figure 12.8). The genome of λ is 48.5kb in length; it was the ﬁrst genome to have its

entire sequence determined (1982). As drawn conventionally, the genome is linear, and

contains 46 genes. At each 5



end of the double-stranded structure is a 12 base single-

stranded sequence known as a cos site; these have a complementary base sequence (i.e.

they are cohesive, or ‘sticky’ ends). They can join together to form a double-stranded,

circular λ molecule, a conformation that is essential for insertion and integration into

the host genome (see Chapter 10).

The naturally occurring phage is unsuitable as a vector, because being relatively

large, it contains multiple copies of recognition sites for a number of REs. By using

genetic manipulation techniques, however, unwanted sites can be removed, allowing

the DNA to be ‘opened up’ at a single location, and fragments of foreign DNA with

complementary sticky ends to be ligated.

A further limitation that must be overcome is that in this form, phage λ can only

accommodate about another 3kb of DNA. This is because if the genome exceeds 52kb,

it cannot be packaged properly into the protein head to produce viable phage particles.

The arrangement of genes on the phage λ genome offers a solution to this problem.

It is known that genes encoding speciﬁc functions are clustered together, with genes

necessary for the lytic replication cycle being found at the ends of the map as it is

conventionally drawn (Figure 12.8). It is possible, therefore, to remove much of the

central part of the λ genome without affecting its ability to infect and lyse its bacterial

host.

Insertion vectors have had some of this non-essential material removed, reducing their

genome size to around 42kb, and thus allowing them to carry an insert of up to 10kb. A

single-copy restriction site is opened up, and a fragment with complementary sticky ends

inserted with the aid of DNA ligase. Vectors such as λZAPII have a multiple cloning site

or polylinker, positioned so that it falls within the lacZ



gene. This allows insertional

Tail Head Recombination cIII N cI cro cII DNA replication Q Lysis

Non-essential genes

Figure 12.8 Bacteriophage λ. The central region of the λ genome contains genes involved

in its lysogenic cycle. This region can therefore be excised, without affecting the ability of

the phage to infect E. coli via the lytic cycle. The genes at the right-hand end of the genome

as shown have a regulatory function, whilst the structural genes are situated at the left

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324 MICROORGANISMS IN GENETIC ENGINEERING

cos cos

BamH1

Cut with

BamH1

BamH1 BamH1

BamH1

Left arm 20 kb

Left arm 20 kb Right arm 9 kb

Left arm 20 kb

Insert

Recombinant

phage DNA

Ligate

Discarded

EMBL4Right arm 9 kb

Right arm 9 kb

Stuffer 14 kb

Figure 12.9 Cloning in bacteriophage λ. The removal of non-essential genes in the centre

of the genome allows the incorporation of large fragments of foreign DNA. From Dale,

JW and von Schantz, M: From Genes to Genomes: Concepts and Applications of DNA

Technology, John Wiley & Sons, 2002. Reproduced by permission of the publishers

inactivation with blue/white selection to be used for the detection of recombinants as

described above for pUC plasmids.

Replacement vectors (Figure 12.9) are able to accommodate larger inserts, because

instead of a single copy of a particular RE site, they have two, one situated either side

of a disposable central section known as the ‘stuffer’ fragment. This can be removed by

digestion with the appropriate RE and replaced by insert DNA. The vector λEMBL3,

which can accommodate inserts of between 9 and 23kb, has two polylinker sequences

containing sites for SalI, BamHI and EcoRI ﬂanking the stuffer fragment, allowing a

variety of restriction fragments to be inserted.

Recombinants in replacement vector systems can be detected by a method that ex-

ploits the limitations in the phage’s ability to package DNA. Just as too big a genome

cannot be packaged (see above), neither can one that is too small (<37kb); consequently,

constructs lacking an insert will not result in the formation of plaques.

Another naturally occurring bacteriophage of E. coli that has been adapted for use

as a cloning vector is phage M13. This is a single-stranded phage that for part of

its replication cycle inside the host cell exists as a double-stranded replicative form,

which can be isolated and manipulated like a plasmid. Systems based on M13 have

proved popular as a means of obtaining DNA in a single-stranded form for applications

such as DNA sequencing using the dideoxy (Sanger) method. M13 vectors contain a

multiple cloning site situated within the lacZ



gene, allowing blue/white selection of

recombinants. The cloning capacity is generally limited to fragments of less than 1.5kb,

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BACTERIOPHAGES AS CLONING VECTORS 325

Box 12.1 A large number of clones are required to cover

an entire genome

The factors that determine the number of clones necessary to make a genomic

library are the size of the genome and the average size of each insert. In addition

the probability that a given fragment will be present in the library must be set: the

higher the probability, the more clones will be necessary.

N =

log(1 − P)

log(1 − a/b)

Where: N = number of clones required; a = average insert size; b = total genome

size; P = probability that a given fragment will be present.

e.g. Number of clones required for a 95% probability of a given fragment to be

present in a genomic library of E. coli (genome size 4.8 × 10

) with an average insert

size of 20kb:

N = log(1 − 0.95)/ log(1 − 4.167 × 10

−3

)

= log 0.05/ log(0.9958)

= 712 clones

This may seem a manageable number, but remember that the genome of E. coli is

very small compared to that of higher organisms (Homo sapiens:3× 10

bp).

but hybrid M13/plasmid vectors (phagemids) have been developed that are able to take

inserts of as much as 10kb.

As we have seen, a large collection of cloned DNA fragments is called a DNA library. If

the original donor DNA comprised the entire genome of an organism, then the collection

of clones, which between them contain an entire donor genome, is known as a genomic

library. We could create a library of the entire E. coli genome (total size 4.8 × 10

bp)

for example, by having just over 700 clones with an average insert size of 20kb (see

Box 12.1 for calculation). For a more complex genome, however, we would either need

to have very many more clones, or to increase the average fragment size.

For this reason, further types of cloning vectors have been developed, to allow the

cloning of larger fragments. Cosmids are entirely man-made creations, and have no

naturally occurring counterpart. They incorporate beneﬁcial features of both plasmids

and phage vectors, and may be capable of accommodating insert fragments of more than

45kb. Cosmids are essentially plasmids that contain the cos sites from λ phage, with

the plasmid supplying the necessary origin of replication, restriction sites and selectable

marker. As we’ve seen, λ DNA will be packaged into phage heads if the cos sites are

37–52kb apart; however the only parts of the DNA recognised by the packaging enzymes

are the cos sites, so any DNA can be used to ﬁll the intervening sequence.

The recombinant DNA is packaged into phage particles by a process called in vitro

packaging, and introduced into the host E. coli. Lacking phage genes, cosmids do not

lead to lysis of the host cells and plaque production, but are instead replicated as if

they were plasmids. Growth of host cells on a selective medium allows the detection of

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326 MICROORGANISMS IN GENETIC ENGINEERING

transformants, i.e. those that have taken up the cosmid. There is no need to select for

recombinants, because non-recombinants are too small to be packaged into the phage

heads.

Expression vectors

Sometimes, the aim of a cloning experiment is not just to obtain large amounts of a

speciﬁc gene, but for the gene to be expressed. This involves using the host cell as a

sort of ‘factory’, to manufacture the speciﬁc protein encoded by the cloned gene. One

of the earliest applications of genetic engineering was the production of human insulin

in E. coli (Figure 12.10). Insulin is needed in considerable quantities for the treat-

ment of diabetics; for years it was obtained from the pancreas of pigs and cattle, but

this had several disadvantages including immunological complications and the risk of

viral contamination. Insulin generated by recombinant means is free from these prob-

lems. Many proteins can now be produced in this way by microorganisms at a rate

several times that of the normal host cell. In order for a gene to be expressed, it must

have speciﬁc nucleotide sequences around it that act as signals for the host cell’s tran-

scription/translation machinery (promoter, ribosomal binding site and terminator – see

Chapter 11). Since these sequences differ between, say, humans and E. coli, the bacte-

rial RNA polymerase will not recognise the human sequences, and therefore, although

a human gene may be cloned in E. coli using a simple vector, it will not be expressed. If,

however, the human gene could be inserted so that it was under the control of the E. coli

expression signals, then transcription should take place. Specially designed vectors that

provide these signals are called expression vectors. The choice of promoter sequence is

particularly important; often, a strong (i.e. very efﬁcient) promoter is selected, so as to

maximise the amount of protein product obtained. Genes whose protein products are

naturally synthesised in abundance are likely to have such promoters. It is often helpful

to be able to regulate gene expression; inducible promoters can be switched on and

off by the presence of certain substances. The lac promoter (which controls the lacZ

gene) is an example of this. Cassette vectors have promoter, ribosomal binding site and

terminator sequences clustered together as a discrete unit, with a single recognition site

for one or more REs being situated downstream of the promoter (Figure 12.11).

The small size of the insulin molecule (and gene) and the size of the potential market

made it a prime early candidate for production by recombinant DNA technology. Most

insulin used in the treatment of diabetes nowadays is produced in this way. Systems

based on E. coli have also been used to synthesise other small human proteins with

The polypeptide pro-

ducts of translation in

eucaryotes may require

post-translational modifi-

cation before functional

protein is produced. Ex-

amples include phos-

phorylation, acetylation

and glycosylation.

therapeutic potential such as human growth hormone,

γ -interferon and tumour necrosis factor (TNF).

Bacteria, however, are not suitable host cells for the

production of many other human proteins such as tis-

sue plasminogen activator (TPA) or blood clotting Fac-

tor VIII, due to the size and complexity of their genes.

This is because many proteins of complex eucaryotes

are subject to post-translational modiﬁcations; this does

not occur in procaryotes, so bacteria such as E. coli are

not equipped with the cellular machinery to make the

necessary modiﬁcations to human proteins.

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EXPRESSION VECTORS 327

Figure 12.10 Production of recombinant human insulin. Human insulin comprises two

short peptide chains, 21 and 30 amino acid residues in length. Because they are so short,

the nucleotide sequence of their genes can be predicted from their amino acid sequence, and

synthetic genes produced. Each is cloned under the inﬂuence of the lac promoter and down-

stream of part of the lacZ



gene. By being produced as fusion proteins with β-galactosidase,

the insulin chains are protected against being degraded by the E. coli cell. From Brown, TA:

Gene Cloning, 3rd edn, Chapman & Hall, 1995. Reproduced by permission of Blackwell

Publishing Ltd.