Genetic Engineering

The Many Purposes of Genetic Engineering

Given that DNA serves as the template for RNA and protein synthesis, the primary objective of genetic engineering is to insert a specific segment of DNA into a microbial host organism with the intention of generating large quantities of a certain RNA or protein product.

In some cases, the downstream products are harvested from the host and purified for medicinal purposes. Quite possibly the most well-known instance of this took place in a 1978 collaborative project involving Genentech and the City of Hope National Medical Center [1]. It was the first laboratory production of human insulin, a protein hormone that regulates blood glucose metabolism and is normally produced in the pancreas. Researchers at Genentech inserted the  gene for human insulin into the genome of Escherichia coli bacteria, which went on to proliferate while produce copious amounts of insulin. Researchers at City of Hope National Medical Center then purified the insulin, separating the protein of interest from their bacterial hosts.

This method served two important advantages:

  1. The procedure was far more cost effective and efficient than the standard procedure of insulin production, which involved the purification of animal insulin from the pancreas of swine and cattle that had been slaughtered for food.
  2. The procedure yielded insulin that was chemically identical to the insulin naturally produced in humans, rather than a slightly dissimilar animal derivative. Consequently, diabetic patients experienced less side effects when given insulin derived from genetically engineered E. coli, rather than when they were given swine or cattle insulin.

In other cases, the of a select group of hosts is modified to serve a benefit in the fields of research, industry, or agriculture. Genetic engineering is typically used in a research setting on bacteria, roundworms, fruit flies, fish, or mice to yield transgenic animals that mimic a certain genetic disease that is seen in humans. Transgenic animals are useful in this capacity for studying the specific pathologies that cause disease or to test the efficacy of therapies that may someday be used in humans. Industrial applications of genetically engineered organisms are extremely diverse and include the production of biofuels, bioremediation (the detoxification of contaminated environments), and production of fermentation products [2,3,4]. The agricultural applications of genetic engineering are similarly diverse and include:

  • The production of produce with an increased shelf life. For example, FLAVR SAVR tomatoes acts by preventing the enzyme polygalacturonase from metabolizing the firmness-inducing pectin [5].

The production of nutritionally enriched foods. For example, Golden Rice 2 produces up to 37 μg/g of the Vitamin A precursor, beta-carotene. The product provides the essential nutrient in regions that are most susceptible to Vitamin A deficiency, a condition that causes the death of an estimated 670,000 children under the age of 5 every year [6,7].

Machinery For Extracting a Gene of Interest

A DNA fragment up to 6 kb that contains a gene of interest can be amplified by a form of cell-free cloning called the polymerase chain reaction (PCR) in preparation for insertion into a target genome. One must generate a mixture of (1) the single-stranded  or denatured DNA fragment that contains a gene of interest, (2) dNTPs, (3) many copies of two oligonucleotide primers whose sequences flank the DNA segment of interest, and (4) heat stable DNA polymerase (Taq DNA polymerase or Pfu DNA polymerase). The mixture is placed into a thermocycler, which cycles through three different temperatures. Each of these temperatures facilitate denaturation (95॰C), annealing, and DNA synthesis. Each cycle produces a doubling of genetic material, such that there exist 2n copies after n number of cycles.

A gene of interest is extracted from the amplified DNA through the application of restriction endonucleases, enzymes that recognize a specific base sequence of 4-8 bases in double-stranded DNA and cleaves both strands of the duplex. There are four different types of endonucleases:

  • Type I: Cleaves DNA at random sites, located at least 100 bp from the recognition sequence.
  • Type III: Cleaves DNA at random sites, located 24-26 bp from the recognition sequence.
  • Type IV: Cleaves and methylated DNA (nucleotides with -CH3 groups appended to their chemical structure.
  • Type II: Cleaves DNA at specific sites within or near the recognition sequence. Their specificity makes them the most useful biochemical tools for genetic engineering.

 

Recognition and Cut Sites of Some Type II Restriction Endonucleases

Enzyme

Recognition Sequence (5’ to 3’)

AluI

AG-CT
BamHI

G-GATCC

Bgl2

A-GATCT
EcoRI

G-AATTC

EcoRV

GAT-ATC

HaeIII

GG-CC
HindIII

A-AGCTT

HpaII

C-CGG
MspI

C-CGG

PstI

CTGCA-G

PvuII

CAG-CTG
SalI

G-TCGAC

TaqI

T-CGA

XhoI

C-TCGAG

*Cut sites are denoted by a hyphen.

 

Many restriction endonucleases catalyze the cleavage of two DNA strands at positions that are symmetrically staged about the center of a palindromic restriction sequence. Catalysis yields restriction fragments with sticky ends – single stranded DNA ends that can associate under annealing conditions by complementary base pairing with other fragments generated by the same restriction enzyme. Once stuck together the two pieces of DNA can be joined covalently through the action of DNA ligase.

DNA fragments with blunt ends must be joined covalently with DNA ligase from bacteriophage T4. Fragments with blunt ends, those that are produced by two different Type II restriction enzymes, and those that are produced by Type I and III restriction enzymes can be joined together by the enzyme terminal deoxynuceotidyl transferase (terminal transferase). Terminal transferase adds nucleotides to the 3’-terminal OH group of a DNA chain. Taking into account the fact that deoxythymine (dT) base pairs with deoxyadenine (dA), artificial sticky ends can be generated by terminal transferase. Poly(dT) tails of approximately 100 residues can be appended to the 3’ ends of fragments containing the gene of interest, while poly(dA) tails of the same length can be appended to the 3’ ends of genome fragments. Although this method eliminates the restriction sites that were used to generate the fragments,  the complementary homopolymer tails will anneal when mixed together and the gene of interest can covalently inserted into the genome by DNA ligase [8].

DNA Cloning Vectors

Typically in genetic engineering genes aren’t inserted into just any old genome, rather into cloning vectors. Cloning vectors are more useful to insert genes into because they replicate independently of their host cell and are often times present in large numbers within that cell. In other words, inserting a gene of Interest into a cloning vector concentrates that gene within the cytosol of less cells. There are several varieties of cloning vectors, but here I will elaborate on four different types: plasmids, bacteriophage lambda, YACs, and BACs [8].

Plasmids

  • DNA Insert Length: < 10 kb
  • Contains the requisite genetic Machinery to permit autonomous propagation in bacterial or yeast.
  • Specifications of plasmids constructed for use in genetic engineering:
    1. Relatively small.
    2. Replicate under relaxed control (independently of the host) and are consequently present in 10-700 copies per cell.
    3. Carry genes specifying resistance to one or more antibiotics. This can be useful in a variety of ways. For instance, if protein synthesis in the host organism is blocked by chloramphenicol, cell division is halted while plasma continue to replicate until 2000-3000  copies have accumulated per cell.
    4. Contain endonuclease sites at which DNA can be inserted.
  • Herbert Boyer and Stanley Cohen (1973) first expressed a chimeric plasmid in a bacterial host. Their bacteria were first coaxed into a state of transformation competence (heating to 42° C, exposure to Ca2+) and, therefore, readily absorb the chimeric plasmids.

Bacteriophage λ

Standard Procedure

  • DNA Insert Length: < 16 kb
  • Insert DNA into the non-essential central third of the viral genome.
  • Introduce DNA to host cells by infecting them with the transgenic bacteriophage λ.
  • Allow host to replicate and collect DNA copies.

Cosmid Procedure

  • DNA Insert Length: < 49 kb
  • Create a cosmid vector by placing two cos sites had a proper distance apart on the viral genome.
  • On introduction to a host cell, cosmid vectors reproduce like plasmids under relaxed control (independent of the host).

Yeast Artificial Chromosomes (YAC)

  • DNA Insert Length: > 49 kb
  • Linear DNA segments, which contain the following self-replication equipment:
    1. Replication Origin: Known as the autonomously replicating sequence (ARS).
    2. Telomeres: The ends of linear chromosomes which preserve the genetic material within the core chromosome.
    3. Centromere: the chromosomal segment that attaches to the spindle during mitosis and meiosis (cell division).

Bacterial Artificial Chromosomes (BAC)

  • DNA Insert Length: > 49 kb
  • Replicate in E. coli bacteria.
  • Are derived from circular plasmids that normally replicate long regions of DNA.
  • Are maintained at a level of one copy per cell.

Genetic Screening

A genetic screening is necessary In order to determine whether or not the gene of interest has been successfully inserted into a cloning vector or whether that cloning vector has successfully entered its host [8]. There are a few types of genetic screens that are typically performed:

Chromogenic (Color-reporting)

A modified gene that encodes the enzyme β-galactosidase, called lacZ’, is inserted into the cloning vector along with the gene of interest. When grown in the presence of the colorless molecule 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal), β-galactosidase hydrolyzes the molecule to produce a blue metabolite. Therefore, colonies of bacteria that (1) have successfully integrated the new genes  and (2) are actively transcribing those genes will form blue colored colonies.  Alternatively, a gene for green fluorescent protein (GFP) can be used as a reporter of transcription since the protein fluoresces under a UV lamp.

Antibiotic Resistance

A gene that confers resistance to the antibiotic ampicillin, called ampR, is inserted into the cloning vector along with the gene of interest. When grown in the presence of ampicillin, bacteria that do not contain the cloning vector perish and those that have successfully taken up the cloning vector will remain.

Southern Blotting

This technique confirms a certain genetic sequence (the gene of interest) is contained within the genetic material of a transformed host. Following digestion of the bacteria and purification of its DNA a gel electrophoresis is performed and the resulting gel is subjected to denaturing conditions, typically by soaking it in 0.5 M NaOH. The basic environment turns the double-stranded DNA into its single-stranded form, one with a high affinity for the nitrocellulose used during blotting.

The DNA from the resulting gel is blotted onto nitrocellulose paper by compressing it onto the gel in a metal clamp and running an electric current through the it while still in NaOH, a process referred to as electroblotting. The blotted nitrocellulose paper is dried so as to fix the DNA is place and is exposed to a minimal quantity of a 32P-labeled single-stranded DNA probe, one that has a complementary sequence to the genetic sequence intended for detection. A phosphorimager is used to visualize the DNA on the nitrocellulose paper, with the labeled DNA appearing as black bands [8].

Genetic Features That Aid in the Production of Protein

One way of coaxing a bacterial host to expressed a trangene that codes for a protein is to place the gene under the control of the lac repressor. Upon exposure to the inducer, isopropylthiogalactoside (IPTG), the lac repressor is deactivated, permitting the expression of the transgenic protein.

However, this strategy is not without its weaknesses. Bacterial cells often sequester large amounts of protein as insoluble and denatured inclusion bodies. To circumvent this difficulty, one can engineer the transgene with a bacterial signal sequence that directs the protein of gram-negative bacteria, like E. coli, to their periplasmic space – a compartment between the plasma membrane and the cell wall.

Once a large quantity of protein has been produced it must be separated from the rest of the bacterial components. To assist in this end, the transgene can be modified to encode a poly-N terminal tail. The poly-N tail has a high affinity to nickel ions and can be purified using a Ni column in a process called affinity chromatography [8].

References

  1. https://www.gene.com/media/press-releases/4160/1978-09-06/first-successful-laboratory-production-o
  2. Summers, Rebecca. “Bacteria churn out first ever petrol-like biofuel.” New Scientist (2013).
  3. Sayler, Gary S., and Steven Ripp. “Field applications of genetically engineered microorganisms for bioremediation processes.” Current Opinion in Biotechnology 11.3 (2000): 286-289.
  4. Dien, B. S., M. A. Cotta, and T. W. Jeffries. “Bacteria engineered for fuel ethanol production: current status.” Applied microbiology and biotechnology 63.3 (2003): 258-266.
  5. Kramer, Matthew G., and Keith Redenbaugh. “Commercialization of a tomato with an antisense polygalacturonase gene: The FLAVR SAVR™ tomato story.” Euphytica 79.3 (1994): 293-297.
  6. Paine, Jacqueline A., et al. “Improving the nutritional value of Golden Rice through increased pro-vitamin A content.” Nature biotechnology 23.4 (2005): 482-487.
  7. Black, Robert E., et al. “Maternal and child undernutrition: global and regional exposures and health consequences.” The lancet 371.9608 (2008): 243-260.
  8. Voet, Donald, and Judith G. Voet. “Biochemistry, 4th Edition.” New York: John Wiley& Sons Inc (2011): 492-496.
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