Monday, November 15, 2021

Applications of Genetic Engineering in Biotechnology

DNA ligase, Applications of Genetic engineering, Recombinant DNA, Molecular DNA scissors, DNA-synthesizer, messenger RNA,

Applications of Genetic Engineering in Biotechnology

The sneaky-snaky messenger RNA

The genetic cope is brought to ribosomes by a messenger! It is called messenger RNA (mRNA). This messenger is both "sneaky and snaky" and carries the same information as the DNA CODE. mRNA resembles DNA, but it is single-stranded Like a "snake". It is slim enough to sneak out of the pores of the nucleus into the cytoplasm. It can escape! 

It has 4 bases, just like DNA. A, C, G, are the same, but T (thymine) in DNA is replaced by a slightly different base called U (uracil). This probably helps all cell enzymes to distinguish RNA from DNA. The "Snaky mRNA" is caught and sandwiched by ribosomes. Its sequence is "read" like a magnetic tape in an old-fashioned tape recorder.

But Ribosomes not only "read" the mRNA, they translate each nucleotide triplet in the genetic code into an amino acid and connect it to the previous one. The resulting long amino acid chain folds into a protein. The DNA sequences in the nucleus that carry the genetic code are called GENES. In genetic engineering, GENES are transferred from one organism to another one, for example the human insulin gene into bacteria.

Plasmids are ideal DNA transporters

Plasmids are DNA rings found in certain bacteria and yeasts. They usually contain 3,000 to 10,000 DNA base pairs, enough to encode 3 to 20 genes. They are hundreds to thousands of times smaller than bacterial chromosomes. Plasmids "live" inside cells independently of chromosomes. They can even move from one bacterium to another via a small bridge, almost like a virus. Antibiotic resistance genes spread this way. This is bad for humans!

Can plasmids be used to carry foreign DNA?

If one could cut open a DNA ring and glue in a foreign gene, the resulting plasmid could bring it inside a cell. It is impossible to achieve this with a mechanical tool. An average gene measures 1/10,000 mm. The diameter of DNA is only 2/1,000,000 mm (2 µm or nanometers). This is incredibly small.

How can one cut something so thin, at the right place?

DNA Ligase - Molecular DNA scissors and glue

Molecular DNA scissors and glue

Bacteria possess molecular scissors called restriction enzymes that can cut DNA inside specific sequences. They were discovered in 1970 by the Swiss microbiologist Werner Arber current President of the Vatican Pontifical Academy of Sciences along with some other smart scientists. The EcoRI restriction enzyme, for example, cuts DNA between G and A, but only within the GAATTC sequence. The opposite DNA strain in the helix is CTTAAG, complementary to GAATTC (remember the Watson-Crick rule!). So, EcoRI cuts both strands, shown as/):



This cut results in two complementary single-stranded DNA ends called "sticky ends": 3’XXXXXXXXG..........AATTCXXXXXXXX-5’


It is possible to glue these sticky ends together with another enzyme called DNA ligase. The discovery of DNA scissors and DNA glue made genetic engineering possible!

Croaking bacteria?

The first genetic engineering experiment took place in early 1973. Stanley Cohen had isolated the plasmid PSC101 (PSC means "plasmid of Stanley Cohen") which carried a gene for resistance against an antibiotic.

This plasmid was first cut open with the EcoRI restriction enzyme, generating a linear segment with sticky ends. Another plasmid, PSC102, conferring resistance against another antibiotic, was cut with the same enzyme.

Cohen's Nobel Prize winning prediction relied on the fact that both plasmid rings pSC101 and PSC102), cut open by the same enzyme, must have the same "sticky DNA ends":



So mixing both linearized plasmids and gluing them with DNA ligase may generate a new, bigger plasmid that did not exist before and that could make recipient bacteria resistant to both antibiotics! The glued plasmids were introduced inside bacteria (remember the Trojan horse! Read this article: Geneticengineering for Human Health - How is Insulin produced - Biotechnology inCartoons) and the two antibiotics were added. Most bacteria died, but a few survived. They contained a new, modified DNA plasmid containing both resistance genes. Recombinant DNA technology was born!

Emboldened by this success, scientists repeated the same experiment using a plasmid and DNA from the African clawed frog. This amazing experiment worked! But the recombinant bacteria did not croak like frogs... Well, perhaps they did, but the sound they made was not loud enough?

Human insulin... from bacteria?

Insulin is essential for life. Some diabetics, affected with type 2 diabetes, require insulin injections every day, all of their lives. The amount a type 1 diabetic requires annually corresponds to the amount found in 50 pig pancreases! The worldwide demand for insulin is incredibly high because of type 2 diabetes. It's a worldwide epidemic: just in the USA, almost 10% of the population have this disease. Type 2 diabetics may still produce some insulin, but most require insulin injections sooner or later.

The structure of insulin was uncovered by Fred Sanger in Oxford, a discovery for which he received the Nobel Prize in 1957. It took him 10 years (starting in 1945) to determine the 51 amino acid sequence of this small protein.

The genetic code being universal, it was possible to deduce the DNA sequence of the human insulin gene. It should contain 51 DNA triplets plus a START and a STOP triplet.

Thanks to genetic engineering, it became possible to "reprogram" bacteria to produce RECOMBINANT human insulin, which is identical to insulin produced by the human organism.

But is genetic engineering really safe?

In 1975, concerned scientists convened in Asilomar, California to assess the potential dangers of Genetic Engineering: "What would happen, if insulin-producing bacteria entered the human body by accident? Would they produce a deadly insulin shock?"

After the Asilomar conference, strict rules were implemented for recombinant DNA experiments, comprising different Safety Levels P1 to P3). So far, there has never been a single accident of any kind due to this technology!

 In 1979, scientists succeeded in building a synthetic insulin gene combining the necessary nucleotides in the lab using a DNA-synthesizer.

Human recombinant insulin produced by microorganisms has been on the market since 1982.

Thank you, BIOTECHNOLOGY, in the name of all diabetics!

Tags: Applications of Genetic engineering, Molecular DNA scissors, DNA ligase, Recombinant DNA, messenger RNA, is genetic engineering safe?, Human insulin... from bacteria.


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