Vectors and PCR

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Vectors and PCR

Viruses and bacteria as vectors:

Viruses and some bacteria are known to transfer some of their DNA or RNA into the cells of a host. This genetic material integrates into the host genome, causing the production of disease or deformity such as Galls.

Galls are large tumour-like growths in plants, usually produced as a response by the plant to some invasion by microbe or insects.

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Characteristics of Agrobacterium:

Agrobacterium tumefaciens is a bacterium, closely related to Rhizobium (found in root nodules and the soil), which has a large plasmid that contains the crown gall disease genes.

It can gain entry to the plant through wounds and cuts. The TI (tumour inducing) plasmid is attracted to the wound site by sugars and other compounds, such as acetosyringone. Even concentrations as low as 10-7M are sufficient to cause attraction.

At higher concentration, the acetosyringone activates the VIR (virulence) genes on the TI plasmid, and these co-ordinate the infection process.

There are two parts to this infective process...

  1. The production of permeases that are inserted into the bacterial cell membrane. These enzymes are for the uptake of compounds (opines) that will be produced by the tumours later.
  2. The production of an endonuclease, which is a restriction enzyme that cuts out part of the TI plasmid, forming a piece of T-DNA (transferring DNA).

The T-DNA is released to enter the host cells where it integrates with the plant DNA and then starts to re-program the whole DNA, so dictating a new function for those cells.

When integrated into the host plant cells, the T-DNA genes are responsible for the production of greater amounts of cytokinin and indole-1-acetic acid (IAA).

These are both plant growth regulators and they will now cause irregular growths of cells, leading to the production of the galls.

Genetic engineering of plants, using Agrobacterium tumefaciens:

Selected genes are engineered into the T-DNA of the bacterial plasmid.

Plants are then infected with the crown gall bacterium, which uses the TI plasmid to incorporate the engineered gene into the plant chromosomes.

Alternatives to this method include firing projectiles (very small pellets), which are coated with the required DNA, into host cells and then growing them on in tissue culture.

The results of these transformations are called transgenic plants.

Examples of commercial uses of transgenic plants:

  1. Tomato 'Flavr Savr' - varieties that can be picked ripe and they do not deteriorate in transit. They do not express a gene for polygalacturonidase, which degrades pectin. As pectin is usually broken down by polygalcturonidase, resulting in softening of the fruit, the absence of this gene action allows the fruit to stay firm and ripe for much longer.
  2. Soya bean 'Roundup Ready' - a glyphosate herbicide resistant variety. When grown in fields, the crop can be sprayed with glyphosate weedkiller, but is not affected, unlike the weeds, which then die.
  3. Cotton, potato and maize - using a similar organism, Bacillus thuringiensis, these plants have been transformed by having a toxin for insect resistance.

How transformation is done:

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The purpose of this PCR is to produce huge numbers of copies of a gene. The enzyme DNA polymerase is added to the sample and when suitably treated, will catalyse many millions of copies of the small sample.

PCR is used as the starting point, or template, for sequencing.

How the PCR works:

There are three steps, repeated for up to 40 cycles in an automatic cycle, which heats and cools the reaction mixture very rapidly.

  1. The DNA strand is heated to 94°C, to denature it and open the strands, forming single strands. All enzyme actions stop.
  2. Annealing at 54°C. During this process the primers are jiggled around by molecular collisions (Brownian motion). Ionic bonds are formed and broken between the single strands of primer and the template. In the areas where more exact fits are made, the bonds last longer, allowing the polymerase enzyme to start copying the template. (The heat stable TaqDNA polymerase comes from a thermophylic bacterium found in hot springs.)

    Note: Very pure DNA building blocks dCTP,dATP,dGTP and gTTP (one for each of the four nucleotide bases) are in the machine at the start.

  3. Extension at 72°C. This is the optimum temperature for the polymerase. Here the bases complementary to the template are coupled to the primer on the 3I side.
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    Because both strands are copied during the PCR process, the rate of increase is exponential.

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When gene transfer is attempted, there must be a way of detecting which microbe has taken up the new gene arrangements. Those that have been unsuccessful have to be eliminated.

One way of identifying successful and failed transformation...

  • At the time when a gene is transferred into the plasmid, another gene for antibiotic resistance is also added somewhere else in the plasmid ring.
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  • The microbes are then transferred to an agar plate containing the selected antibiotic, and incubated for 24 hours.
  • After incubation, colonies are seen of the agar. These will be the transformed microbes that have the desired gene. They have survived because of the antibiotic resistance marker gene.
  • Samples of these colonies are used to inoculate further liquid cultures, to build up huge numbers of the transformed microbe.

Another way of identifying a modified organism is to attach a fluorescent marker, which can be seen using fluorescent microscopy techniques.