INTRODUCTION: Molecular cloning in biotechnology is rapidly becoming a major force in

research. A key step of gene cloning is to recombine a gene of interest into a plasmid vector.

Ideally, a gene is isolated on a restriction fragment created by two different endonucleases that

cleave on either side of the gene and that generate distinctive single-stranded ends. The sticky

ends (each strand cleaved off-center in the recognition site, at positions two to four nucleotides

apart) contain fragments with exposed ends of short, single-stranded sequences. The particular

enzyme used here was EcoRI, which leaves 5’overhangs of four nucleotides. The sticky ends

of the restriction fragment are then rejoined to the complementary ends of a plasmid vector that

has been opened up with the same two enzymes. Such "directional cloning" using two different

enzymes produces restriction fragments that have noncomplementary overhangs at each end. This  prevents any fragment from rejoining its own ends and encourages recombination between different

fragments.  The single-stranded overhang of a sticky end can form hydrogen bonds with the 

enzyme.  Hydrogen bonding of several nucleotides is not sufficient to form a stable molecule, so

associations between complementary ends constantly form and break.  This transient inter-

action, however, does not hold the two restriction fragments together long enough for DNA ligase

to re-form phosphodiester bonds between adjacent nucleotides.  This covalently links the de-

oxyribose-phosphate rails of the two fragments into a stable double helix.  During the ligation

reaction, an ester linkage is formed between the terminal phosphate of the 5' overhang of one frag-

ment and adjacent deoxyribose ring at the 3' complementary nucleotides in the overhang fragment

of another fragment generated by the same restriction end of the second fragment.  This is

accompanied by the loss of one molecule of water, making ligation an example of a condensation

reaction.

In this laboratory exercise, a newly genetically engineered plasmid, pCR4-

TOPO, was used, which requires only one endonuclease (EcoRI). There are two recognition site

for this enzyme located on either side of the insertion area, which allows the relatively easy

insertion of the hsp16.6 gene, which was isolated in lab #1, and amplified and electrophoresed

in lab #3. This special plasmid also contains genes which allows for selection of the clone

three different ways: 1) successful insertion of the desired gene, hsp16.6, interrupts an operon, which,

when not interrupted, allows for metabolism of the chemical X-gal, which has been added to the

growth medium for the host bacterium. The recombinant bacteria will not metabolize X-gal and

will be white in color. If they had not had the insert, metabolism would have turned the bacterial

colonies blue. 2) There are also genes for ampicillin and kanamycin resistance located in the plas-

mid away from the insertion area, so successful recombination results in bacteria which are resis-

tant to both antibiotics if placed in the growth medium; and 3) the vector contains the lethal ccdB gene

fused to the C-terminus of the LacZa fragment. Ligation of a PCR product disrupts expression of the

lacZa-ccdB gene fusion permitting growth of only positive recombinants upon transformation in TOP10 

cell.  Cells that contain non-recombinant vector are killed upon plating.

                The students will follow protocol and perform the cloning reaction, which will result in

the chemical transformation of E.coli bacterium. They will hopefully then be able to explain the importance

of each major step in the ligation and transformation.

MATERIALS AND METHODS: See the following pages for protocol and note any

modifications.

        To be added.

RESULTS: The growth of many colonies of E.coli on the incubated medium was evident. There

were both blue and white colonies.

DISCUSSION: Successful recombination of the plasmid was evidenced by the white colonies

which probably had taken up the hsp 16.6 gene.  If there were blue colonies, there was no

interrupted metabolism of the X-gal in the medium.  By virtue of their survival, the ccdB gene

mentioned in the discussion had been prevented from being expressed in the bacteria.  Survival

in the ampicillin medium was not a factor here, because the location of the insertion of the gene into

the plasmid was away from the gene for ampicillin resistance and had not prevented its

expression. Further analysis of these bacteria in lab #5 will be done, to further prove or

disprove the evidence of successful recombination. The plasmid will be subjected to restriction

enzyme digestion and the digested fragments will be electrophoresed and sized to determine if

the gene in question shows up as a 451 bp fragment in the gel.

    If this lab were to be done in a high school classroom, the following questions may help students

understand the biotechnology here:

1. How efficient is colony transformation? "DNA Science," a text by David Miklos and

Greg Freyer, Cold Spring Harbor Press, 1990, may be helpful in addressing this concept.

2. What further experiments may be done to prove the success of the transformation?

3. What are the ramifications of this type of laboratory work?

4. How may this knowledge and technology affect our society?

CONCLUSION: The ability of researchers to select a gene and successfully separate it from the

chromosomal DNA and then transfer it to a well-studied foreign host cell is both encouraging

and frightening. Genetic material introduced into foreign cells is replicated and passed on to

progeny cells. Today many methods are available for isolating and characterizing genes and

proteins.  Selected genes are being transferred to organisms such as plants, animals,

bacteria, and fungi for a variety of reasons: Commercially desirable products can be efficiently

produced in host cells, genes and their proteins can be studied in ways not possible before,

and medical biotechnologies are being explored. It is imperative that our young students be

exposed to the techniques and research in this rapidly changing biotechnological age. They

are in the midst of a "paradigm shift" which has impacted society already, and will only

continue to do so. Along with these advances, the burden for responsible behavior is

tremendous, and the youth will have a heavy load to carry.

 

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