Bacteria Transformation

• Bacteria are useful instruments for gene study because they can be induced (coerced) into taking up foreign DNA (transformation) for later expression.

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

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

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

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

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