lec45gmo - Control of Gene Expression Control Promoter...

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Unformatted text preview: Control of Gene Expression Control Promoter regions can determine whether or not a Promoter gene is activated gene Cis elements and Trans factors Cis elements- regions of DNA to which trans regions factors bind factors Regulatory genes code for trans factors that Regulatory activate other genes activate Promoters can be constituative- always on constituative Promoters can become active at stages of Promoters development development Can become active under certain environmental Can conditions conditions Can be useful for genetic engineering Transforming an Organism Transforming Find your gene of interest Find Insert it into genome of organism Insert Regenerate organism Regenerate Determine if gene is functional Determine Genetic Engineering Genetic Find your Gene of Interest Protein/mRNA—reverse transcriptase and Protein/mRNA reverse cDNA cDNA Reverse Transcriptase- viral enzyme makes Reverse viral DNA from RNA-called cDNA DNA called cDNA Related Gene/ from other organism Related Find your gene in a library Find Make a Library Make Library- collection of fragments of genome Library collection Genomic Library—Restriction enzymes Genomic cDNA Library cDNA Insert genes into Plasmids and grow in bacteria Insert Find your gene of interest in fragments Find Clone it in bacteria- make lots of copies Clone Restriction Enzymes Restriction Cut DNA with sticky ends- useful for splicing Cut useful fragments together fragments Genomic Library Genomic Cut DNA into lots of pieces Cut Insert Pieces into plasmids Insert Insert plasmids into Bacteria Insert Grow Bacteria and Multiply fragments Grow Probe cloned fragments to find your gene Probe Bacterial Genome Bacterial Bacteria have a single circular chromosome Bacteria Also contain plasmids- small circular DNA Also small which are transferred during conjugation-genetic which genetic recombination recombination Once you have your gene reinsert into Bacteria and make lots of copies and Use a reporter gene to identify recombinant colonies colonies Usually antibiotic resistance or a reporter gene Usually Transforming an Organism Transforming Find your gene of interest- Buy it or make a Find Buy genomic library and find it genomic Insert it into genome of organism Insert Regenerate organism Regenerate Determine if gene is functional Determine OK, I found my Gene, Now what? OK, Make lots and lots of copies- amplification Make Reinsert into Bacteria and grow up cultures Reinsert Need a marker to identify colonies with your gene- antibiotic resistance genes gene Grow culture with antibiotic- only resistant Grow only cultures will grow that have the genes cultures Remove your gene with restriction enzymes Remove PCR PCR Polymerase Chain Reaction- another method to another amplify your gene amplify Uses a special DNA polymerase which works at Uses high temperatures to multiply DNA quickly without Bacteria- Taq Polymerase without No marker needed needed Insert your gene into your organism of Interest Interest For plants need to deal with cell wall, animal systems only cell membrane systems Promoters/ Reporter genes Promoters/ Need some type of marker to identify transformed cells- antibiotic resistance or marker transformed antibiotic genes genes Green Fluorescent Protein Green Insert your gene into your organism of Interest Interest Bacterial systems—Protein/enzyme production Bacterial Electroporation/ChemoPoration/lipisomes Electroporation/ChemoPoration/lipisomes Biolistics- gene gun Biolistics Microinjection Microinjection Retroviruses—mRNA Retroviruses Agrobacterium tumefaciens –Plant System for Agrobacterium Plant transformation transformation Electroporation/Chemoporation Electroporation/Chemoporation Use electric current or chemicals to make the Use membrane permeable for your gene to be incorporated into cell/nucleus incorporated Insert your gene into your organism of Interest Interest Bacterial systems—Protein/enzyme production Bacterial Electroporation/ChemoPoration/lipisomes Electroporation/ChemoPoration/lipisomes Biolistics- gene gun Biolistics Microinjection Microinjection Retroviruses—mRNA Retroviruses Agrobacterium tumefaciens –Plant System for Agrobacterium Plant transformation transformation Biolistics Biolistics Attach copies of gene to small particles and Attach “shoot” them into cells Gene gun- Biolistics Gene Insert your gene into your organism of Interest Interest Bacterial systems—Protein/enzyme production Bacterial Electroporation/ChemoPoration/lipisomes Electroporation/ChemoPoration/lipisomes Biolistics- gene gun Biolistics Microinjection Microinjection Retroviruses—mRNA Retroviruses Agrobacterium tumefaciens –Plant System for Agrobacterium Plant transformation transformation Agrobacterium tumefaciens Agrobacterium Pathogenic bacterium that infects plant tissue and induces tumors and Incorporates portions of its genome into the plant genome- causes uncontrolled growth of plant causes infected cells infected Desirable genes can be inserted using nonDesirable virulent strains After Transformation After Grow up cells and identify transformantstransformants reporter gene or antibiotic resistance Regenerate organism from transformed cells Regenerate Tissue culture in plant systems Tissue If interested in gene product, then culture trasformed cells, extract product trasformed Concerns Concerns Insertion is random in genome Insertion Marker or reporter genes are present in transformed organism- antibiotic resistance transformed ...
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