What is Transformation?. Change… In molecular biology, change in which genetic material carried by an individual cell is altered by incorporation of foreign DNA. Objective 1 - Define the term plasmid and explain its significance for bacteria and recombinant technology.
In molecular biology, change in which genetic material carried by an individual cell is altered by incorporation of foreign DNA
A plasmid is a small piece of circular DNA in bacteria in addition to bacterial chromosome
Typically contain genes for traits beneficial for survival such as antibiotic resistance
In nature, bacteria transfer these plasmids back and forth
Acts as a vector – a gene taxi to get new DNA into bacteria
How is DNA isolated?
Also called restriction endonucleases
1962: “molecular scissors” discovered in in bacteria
E. coli bacteria have an enzymatic immune system that recognizes and destroys foreign DNA
3,000 enzymes have been identified, around 200 have unique properties, many are purified and available commercially
“Able was I, ere, I saw Elba” restriction enzymes is useful in producing a recombinant DNA molecule.
Bam H1 site:Restriction Endonucleases
Recognition sites have symmetry (palindromic)
Enzymes recognize specific 4-8 bp sequences
Some enzymes cut in a staggered fashion - “sticky ends”
Some enzymes cut in a direct fashion – “blunt ends”
Complementary base pairing restriction enzymes?
+ DNA Ligase, + rATP
recombinant DNA molecule
Sticky end restriction enzymes in transformation
Human DNA cleaved with EcoRICorn DNA cleaved with EcoRI
Restriction enzyme animation restriction enzymes?
Go to Techniques – cutting and pasting
Recombinant restriction enzymes?
E. coli host cell
Objective 4 - Explain each of the steps taken to isolate a gene of interest and insert it into bacterial DNA to produce the desired protein.
The genomic DNA and a bacterial plasmid are isolated
Both are digested with the same restriction enzyme
The fragments are mixed, and DNA ligase is added to bond the fragment sticky ends
The solution with the transformed plasmid is added to bacteria.
Bacteria take up plasmid
Bacteria are grown in culture dishes, creating “clones” that should all have the plasmid in them
But how do we check?
A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene
The plasmid may also incorporate an ampicillin resistance gene, then the bacteria grown in the presence of ampicillin, only the bacteria with the plasmid of interest should survive.
A gene for fluorescent protein may be incorporated into the plasmid with the gene of interest, showing which organisms now carry the gene of interest
Ampicillin Resistance Gene transformation has been successful.
Bioluminescent AND antibiotic resistant
Wash cells from plates and centrifuge, discard liquid
Lyse bacterial cells in a non-denaturing environment (protease inhibitors and buffer solution)
Centrifuge and collect the liquid for soluble proteins and the pellet for non soluble protein
Check with gel electrophoresis
Information from (among others) microvet.arizona.edu/Courses/MIC205/10GenEng/GeneticEng
Ineering and www.udel.edu/chem/bahnson/chem527