13-3 Cell Transformation (Changes in Cell’s DNA)
Review• Which enzyme is used as a scissor in genetic engineering ? Which one is used as glue? Sciessor: Restriction Enzyme Glue: DNA ligase• What’s the function of gel electrophoresis? Separate DNA fragments by their different size
13-3 Cell transformation• Cell transformation= change the cell’s DNA• A cell takes DNA from outside. The external DNA becomes part of the cell’s DNA
1) Transforming bacteria cells – Using plasmid • small circle of extra bacterial DNA, double strand • Foreign DNA can be inserted into plasmid – Forms recombinant plasmids – Plasmids used in genetic engineering are called vectors. – Can deliver DNA into another cell
1) Transforming bacteria cells – Using plasmid • Plasmid has a origin site for replication • Plasmid has a genetic marker (usually a gene for resistance to antibiotics) • two types of plasmid integration into a host bacteria
Restriction enzyme DNA or cDNA Foreign DNA, plasmidcuts molecule fragments with DNA, and modificationof chromosomal DNA sticky ends enzymes are mixedor cDNA recombinant plasmids containing foreign DNASame enzyme cuts plasmid DNAsame sequence in with sticky endsplasmid DNA host cells containing recombinant plasmids
What if insertion eukaryotic DNAinto bacteria ?Will there be any problems ???
Transcription RNA polymerase mRNA splicingDNA Pre-RNA mRNA No introns Bacteria don’t know how to deal with introns USE cDNA!!!
Reverse transcription= Making cDNA mRNA transcript Reverse transcriptasecDNA -- A DNA reversetranscription from a mRNA–cDNA hybridmatured mRNA `“c” -- complementary single-stranded cDNA DNA polymerase double-stranded cDNA
2) Transforming plant cells• Recombinant plasmid is transformed to plant – By using bacterium Agropbacterium tumefaciens – By gene gun injection
By using Agrobacterium Plasmids carrying the gene of interest is transferred to agrobacterium The agrobacterium inserts the foreign DNA into the plant cell to form transgenic cell
By using gene gun To coat metal particles with gene containing plasmids Gene gun injects the DNA covered particles into the plant cell
3) Transforming animal cells• Animal cell – Inject into egg nucleus some enzymes may help to insert foreign DNA into chromosomes – Gene replacement ( gene “knockout”)