Bacteria Transformation Bacteria are useful instruments for gene study because they can be induced...
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Transcript of Bacteria Transformation Bacteria are useful instruments for gene study because they can be induced...
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Bacteria Transformation
• Bacteria are useful instruments for gene study because they can be induced (coerced) into taking up foreign DNA (transformation) for later expression.
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Adding DNA to E-Coli• Most techniques for cloning make use of bacteria (E. coli) as they have small circular plasmids that are receptive for insertion of foreign DNA.– Plasmid is isolated from the
bacteria • the plasmid useful for may already
be prepared with an insertion site (restriction site) and an enzyme that infers antibiotic resistance (ampR)
– restriction enzymes are used to isolate the gene of interest and to open the plasmid
• DNA is attached to the plasmid, at the sticky ends using DNA ligase – DNA containing the gene of
interest is mixed with the open plasmids so that the sticky ends can attach
– DNA ligase is added after to permanently join the segments
– now called the plasmid cloning vector
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Confirmation of Transformation• Plasmid DNA with the gene inserted
is now known as recombinant DNA – Plasmid is inserted back into the
bacteria via transformation and grown on special agar containing an antibiotic or color marker
– Competent (transformed bacteria) are harvested and grown on new plates• The bacteria produces clones of itself
through binary fission– The products can then be harvested
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YAC• Yeast cells also make good cloning
vectors – contain a larger plasmid – are single celled eukaryotic
organisms – can incorporate YACs (yeast
artificial chromosomes) into the organism • reproduce by mitosis and contain
their own promoter regions • allow for even larger DNA pieces to
be added
• DNA can be incorporated into animal cells through the process of electroporation – electrical impulses that poke holes
in the plasma membrane
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Knock-out genes• Genes are inserted into a eukaryotic egg– Similar sequence as
known gene– Contains a marker for
study– Allows the researcher to
see affect that the absent product has on the organism
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DNA Libraries• Cloned DNA can be stored in DNA libraries for future use and study
– can be packaged in bacteriophages (phage vector) for quick insertion and cloning bacteria plasmids
– a complete set of phage clones is called a genomic library – an advantage is that larger DNA particles can be incorporated in the phage vector
than in the plasmid DNA • DNA libraries can also be created from the mRNA transcribed from the genes
- called cDNA libraries – reverse transcriptase is used to make the complimentary DNA strand – the cDNA is then inserted into a vector and stored – an advantage is the cDNA contains only exons as the mRNA is already modified from
the original transcript