Lab Notes and Procedures-11 Biology 1004

North Arkansas College

Topic: Molecular Genetics

Produce a Strain of E. Coli Bacteria that Glows in the Dark

  1. E. coli is a bacterium normally found in the intestinal tract (usually not a pathogen). It does not normally glow in the dark.
  2. Many bacteria contain plasmids. Plasmids are small segments of DNA found in cytoplasm à not part of chromosome. Plasmids carry 5-100 different genes.
  3. Most plasmids can be "cut open" with specific enzymes called restriction endonucleases. These are enzymes that recognize short sequences (4-7 bases) that run in reverse on the 2 strands.

- G A A T T C -

- C T T A A G -

(restriction enzymes)

  1. These enzymes "open up" circular DNA (plasmid). By cutting DNA from foreign sources, we can clone or incorporate fragments of this foreign DNA into the plasmid. This is the foundation of cloning. We will use a plasmid called PUC-18.
  2. Some bacteria can "take up" smaller amounts of free or naked DNA by a process called transformation. The bacteria must be competent to take up the free DNA.
  3. These bacteria can be made competent by placing them in calcium chloride in an ice bath. This increases the size of the pores in the cell wall/membrane.
  4. Bacteria are normally susceptible to certain antibiotics. An antibiotic is a chemotherapeutic agent that is produced by 1 organism that has the ability to kill other organisms. Penicillin makes up a group of commonly used antibiotics. The natural Penicillins are active against only a few organisms (mostly G+), but some newer semi-synthetic Penicillins, such as Ampicillin, are active against a wide variety of bacteria.
  5. Many bacteria develop resistance to antibiotics. This resistance can be used to select those bacteria that carry our plasmid (PUC-18). If Ampicillin resistance gene is placed into this plasmid, it can grow on media that contains Ampicillin.

Some living organisms (firefly's, fish and bacteria) are bio-luminescent (glow in dark). This is a protein/enzyme (luciferan/luciferase). The genes that control this bio-luminescence lie in the part of genome called the Lux Operon Gene's A & B from photo bacterium fischeri.

Mendelian Genetics: Simple Monohybrid Cross

Cross pure parents for a particular strain. The first offspring (F1) - all of F1 will be like 1 parent, and F2 will produce 3 offspring like 1 parent, and ¼ of offspring like other parent. This occurs if the genes are dominant and recessive.

Homozygous - both alleles are same (AA).

Heterozygous - both alleles are different (Aa).

When you have homozygous dominant and homozygous recessive (aa) mate - all of F1 offspring will be Aa.

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