genetic-engineering-promoters-and-marker-genes

Genetic engineering: promoter

• The promoter is the region of DNA that determines which gene will be expressed

• This is because it is the site where RNA polymerase binds in order to begin transcription

  • A promoter is one example of a length of non-coding DNA that has a specific function

• The promoter also ensures that RNA polymerase can recognise which strand is the DNA template strand

  • RNA polymerase recognises the template strand as the promoter contains the transcription start point (the first nucleotide of the gene to be transcribed) which is where the enzyme will bind

• Thus the promoter is used to regulate gene expression because only if it is present will transcription and therefore the expression of the gene occur

• If genetic engineers want to ensure the desired gene is expressed when modifying the plasmid they have to add an appropriate promoter

• As with eukaryotic cells, bacteria have many different genes coding for many different proteins although not all genes are switched on at once

• Bacteria will only express genes (to make proteins) if the growing conditions require a certain protein (e.g., E. coli bacteria only make β-galactosidase enzymes when their growing medium contains lactose but lacks glucose)

• Scientists used this knowledge when first genetically engineering bacteria to produce insulin

  • In this case, they added the insulin gene along with the β-galactosidase gene to share a promoter (which switched on the gene when the bacteria needed to metabolise lactose)

  • So when the scientists grew the bacteria in a medium containing lactose but no glucose, the bacteria produced the β-galactosidase and human insulin

Genetic engineering: marker genes

• A marker is a gene that is transferred with the desired gene to enable scientists to identify which cells have been successfully altered and now contain recombinant DNA

• Antibiotic-resistant genes were once commonly used as marker genes

  • Scientists genetically modified the bacteria so that the plasmid contained the desired gene along with a specific antibiotic-resistant gene (and promoter) and then grew the bacteria on agar plates embedded with that antibiotic

  • The bacteria that contained the recombinant plasmids could be identified as these were the bacteria that grew successfully

• Using antibiotic-resistant genes as marker genes concerns scientists as:

  • There is a risk that the antibiotic-resistant genes could be accidentally transferred to other bacteria including pathogenic strains creating pathogenic antibiotic-resistant bacteria

  • If the resistance spreads to other bacteria this could make antibiotics less effective

• The spread of antibiotic-resistant genes can occur due to:

  • conjugation (the transfer of genetic material from one bacterium to another), or

  • transduction (the transfer of genetic material from one bacterium to another via a virus)

• So genes that express proteins that are fluorescent are now commonly used as markers

• The fluorescence is due to the presence of a green fluorescent protein (GFP)

• The GFP gene along with the desired gene are linked to a specific promoter

• Once this promoter is activated, and the protein is expressed, the recombinant bacteria are detected when they glow green under exposure to ultraviolet light

• The use of fluorescent genes as markers is preferable because:

  • They are easier to identify (all that is required is the ultraviolet light)

  • More economical (do not need to grow the bacteria on plates of agar infused with antibiotics)

  • No risk of antibiotic resistance being passed on to other bacteria

  • There are antibiotics that are no longer effective and therefore would not stop any bacteria from growing