Detecting Insertions in Vectors

Inserts in a vector can be checked by isolating DNA, cutting with a restriction enzyme, and seeing how many fragments are generated.

Inserts are sometimes screened by the change in growth properties due to disrupting a gene on the vector.

Inserts are often detected by blue and white screening with Xgal. Inserts abolish production of beta-galactosidase and result in white (rather than blue) colonies.

Once a gene or other fragment of DNA has been cloned into a plasmid vector and transformed into a bacterial cell, we face the problem of detecting its presence. The plasmid itself may be detected by conferring antibiotic resistance on the host cell, but this leaves the question of whether the presumed insert is actually there. If the cloned gene itself codes for a product that is easy to detect, there is no problem. In most cases, however, the presence of the inserted DNA itself must be directly monitored.

The least sophisticated and most tedious method is to screen a large number of suspects for the inserted DNA. Many separate bacterial colonies that received the plasmid vector, hopefully with DNA inserted, are grown in separate vials. Plasmid DNA is extracted from each of these bacterial cultures and cut with the restriction enzyme used in the original cloning experiment. If there is no insert in the plasmid, this merely converts the plasmid from a circular to a linear molecule of DNA. If the vector contains inserted DNA, two pieces of DNA are produced, one being the original plasmid and the other the inserted DNA fragment. To see how many fragments of DNA are present, the cut DNA is separated by agarose gel electrophoresis. If enough transformed colonies are tested, sooner or later one carrying a plasmid with the inserted DNA fragment will be found. This approach was of necessity used in the early days of genetic engineering. Today, modified vectors are available that facilitate screening by a variety of approaches.

Rather less laborious is to use a plasmid with two antibiotic resistance genes. One antibiotic resistance gene is used to select for cells, which have received the plasmid vector itself. The second is used for the insertion and detection of cloned DNA (Fig. 22.14). The cut site for the restriction enzyme used must lie within this second antibiotic resistance gene. When the cloned fragment of DNA is inserted this antibiotic resistance gene will be disrupted. This is referred to as insertional inactivation. Consequently, cells that receive a plasmid without an insert will be resistant to both antibiotics. Those receiving a plasmid with an insert will be resistant to only the first antibiotic.

The most convenient and widely used method to screen for inserts uses color screening.The most common procedure uses b-galactosidase and X-gal to produce bacterial colonies that change color when an insert is present within the vector. The process, called blue/white screening, has a unique vector that carries the 5'-end of the lacZ gene. This truncated gene encodes the alpha fragment of b-galactosidase, which consists of the N-terminal region or first 146 amino acids. A specialized bacterial host strain is required whose chromosome carries a lacZ gene missing the front portion but encodes the rest of the b-galactosidase protein. If the plasmid and chromosomal gene segments are active they produce two protein fragments that associate to give an active enzyme. This is referred to as alpha complementation (Fig. 22.15). Note that assem-

alpha complementation Assembly of functional b-galactosidase from N-terminal alpha fragment plus rest of protein alpha fragment N-terminal fragment of b-galactosidase beta-galactosidase (b-galactosidase) Enzyme that cleaves lactose and other b-galactosides so releasing galactose blue/white screening Screening procedure based on insertional inactivation of the gene for b-galactosidase insertional inactivation Inactivation of a gene by inserting a foreign segment of DNA into the middle of the coding sequence X-gal Chromogenic substrate that is split by b-galactosidase so releasing an insoluble blue dye

Restriction

Restriction

Allow plasmid to replicate-occasional mutations will occur
Cut with restriction enzyme

Transform into bacteria

Only mutant plasmid survives

Only mutant plasmid survives

FIGURE 22.13 Eliminating Unwanted Restriction Sites

The restriction site shown in blue is unwanted. During normal DNA replication, occasional mutations occur. Consequently a very small percentage of the plasmids will carry a random mutation (red) that alters this particular restriction recognition sequence. A sample of the plasmid DNA is isolated from a bacterial culture. The plasmids are treated with the appropriate restriction enzyme. All will be cut, except those with mutant restriction sites. The mixture is then transformed back into bacterial cells. Bacteria receiving cut, linearized plasmids will degrade them. Only mutant plasmids which remain circular will survive.

FIGURE 22.14 Screening for Insert by Disruption of Antibiotic Resistance

A plasmid that has a unique restriction enzyme site within an antibiotic resistance gene can be used to identify those plasmids into which a cloned gene has been inserted successfully. If the gene of interest is ligated into this restriction site, the antibiotic resistance gene will no longer be active. Any bacteria harboring the plasmid with an insert will no longer be resistant to this particular antibiotic.

Antibiotic resistance gene #1

Lígate insert into

Vector resistance gene #2

FIGURE 22.14 Screening for Insert by Disruption of Antibiotic Resistance

A plasmid that has a unique restriction enzyme site within an antibiotic resistance gene can be used to identify those plasmids into which a cloned gene has been inserted successfully. If the gene of interest is ligated into this restriction site, the antibiotic resistance gene will no longer be active. Any bacteria harboring the plasmid with an insert will no longer be resistant to this particular antibiotic.

Antibiotic resistance gene #2

Cut site

Antibiotic resistance gene #2

Lígate insert into

Vector resistance gene #2

Insert

Cells carrying vector are resistant to both antibiotics

Cells carrying vector with insert are resistant to first antibiotic only

FIGURE 22.15 Alpha complementation

The ß-galactosidase protein is unique since it can be expressed as two pieces that come together to form a functional protein. The two protein fragments can be encoded on two different molecules of DNA within the bacterial cell. The alpha fragment can be expressed from a plasmid and the remainder of the ß-galactosidase can be expressed from the chromosome.

>cza

>cza

Transcription and translation

Alpha fragment

Rest of LacZ protein

Assembly

Assembly

Active ß - galactosidase

Active ß - galactosidase

Alpha fragment

Rest of LacZ protein bling an active protein from fragments made separately is normally not possible. Fortunately, ß-galactosidase is exceptional in this respect. The reason for splitting lacZ between plasmid and host is that the lacZ gene is unusually large (approximately 3000 bp—almost as large as many small plasmids) and it greatly helps if cloning plasmids are small.

In order to utilize this unique protein for cloning, a polylinker is inserted into the lacZa coding sequence on the plasmid, very close to the front of the gene. Luckily, the very front most part of the ß-galactosidase protein is inessential for enzyme activity. As long as the polylinker is inserted without disrupting the reading frame, the small addition does not affect the enzyme. However, if a foreign segment of DNA is inserted into the polylinker, the alpha fragment of ß-galactosidase is disrupted and no active enzyme can form (Fig. 22.16). The active form of ß-galactosidase splits X-gal, which produces a blue color (see Ch. 7 for details). Plasmids without a DNA insert will produce ß-galactosidase and the bacterial cell that carries them will turn blue. Plas-mids with an insert will be unable to make ß-galactosidase and the cells will stay white.

Multiple cloning site

Plasmid DNA íacZa MCS íacZa

FIGURE 22.16 Blue/White Screening for p-Galactosidase

In order to screen for inserts in a plasmid with the lacZa gene, a small polylinker is inserted into the extreme N-terminal portion of lacZa. The small insertion is cloned in-frame, therefore, the alpha fragment is still active when complexed with the remainder of p-galactosidase expressed from the chromosomal gene. Bacteria containing this construct will turn blue in the presence of X-gal. However, if a large segment of DNA, such as a cloned gene, is inserted into the multiple cloning site, the alpha fragment is disrupted and p-galactosidase is no longer active. Bacteria harboring this plasmid cannot cleave X-gal, and therefore remain white.

protein synthesis dna is ligated into multiple cloning site

Cells form blue colonies lacZa MCS Inserted DNA MCS lacZa ■ _ _

protein synthesis

Defective front part of b - galactosidase

Cells form white colonies

Active front part of b - galactosidase

Specialized vectors have been made that can replicate in more than one organism. This allows the same gene to be expressed in different hosts.

Shuttle vectors must have separate origins of replication and separate selection mechanisms for each host organism.

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