Making Cells Glow: Bacterial Transformation with pGLO Plasmid DNA
Bacterial Transformation RET Summer 2007. Overall Picture Bio-Rad pGLO Transformation Insertion of...
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![Page 1: Bacterial Transformation RET Summer 2007. Overall Picture Bio-Rad pGLO Transformation Insertion of GFP gene into HB101 E. coli.](https://reader035.fdocuments.in/reader035/viewer/2022072009/56649d8e5503460f94a765ee/html5/thumbnails/1.jpg)
Bacterial Transformation
RET Summer 2007
![Page 2: Bacterial Transformation RET Summer 2007. Overall Picture Bio-Rad pGLO Transformation Insertion of GFP gene into HB101 E. coli.](https://reader035.fdocuments.in/reader035/viewer/2022072009/56649d8e5503460f94a765ee/html5/thumbnails/2.jpg)
Overall Picture
Bio-Rad pGLO TransformationInsertion of GFP gene into HB101 E. coli
![Page 3: Bacterial Transformation RET Summer 2007. Overall Picture Bio-Rad pGLO Transformation Insertion of GFP gene into HB101 E. coli.](https://reader035.fdocuments.in/reader035/viewer/2022072009/56649d8e5503460f94a765ee/html5/thumbnails/3.jpg)
Transformation• The process of transferring foreign DNA
fragments into a recipient (host) cell for growth and replication
• Our host cells: HB101 E. coli
• Our foreign DNA: GFP & -lactamase genes (contained in the pGLO plasmid)
![Page 4: Bacterial Transformation RET Summer 2007. Overall Picture Bio-Rad pGLO Transformation Insertion of GFP gene into HB101 E. coli.](https://reader035.fdocuments.in/reader035/viewer/2022072009/56649d8e5503460f94a765ee/html5/thumbnails/4.jpg)
Plasmids• Plasmids
– small (1-1000 kb)– circular– extrachromosomal DNA
• Growth is independent of the host’s cell cycle; amplification of gene product
• A type of cloning vector used to carry a gene not found in the bacterial host’s chromosome
![Page 5: Bacterial Transformation RET Summer 2007. Overall Picture Bio-Rad pGLO Transformation Insertion of GFP gene into HB101 E. coli.](https://reader035.fdocuments.in/reader035/viewer/2022072009/56649d8e5503460f94a765ee/html5/thumbnails/5.jpg)
Overall Transformation Process
1. The plasmid vector must be cut with a restriction endonuclease (aka: restriction enzyme)
2. DNA ligase joins the DNA fragment & vector DNA
3. Host cell is made competent so can plasmid can enter
4. Transformed cells are grown on selection media
![Page 6: Bacterial Transformation RET Summer 2007. Overall Picture Bio-Rad pGLO Transformation Insertion of GFP gene into HB101 E. coli.](https://reader035.fdocuments.in/reader035/viewer/2022072009/56649d8e5503460f94a765ee/html5/thumbnails/6.jpg)
Overall Transformation Process
1. The plasmid vector must be cut with a restriction endonuclease (aka: restriction enzyme)
2. DNA ligase joins the DNA fragment & vector DNA
3. Host cell is made competent so can plasmid can enter
4. Transformed cells are grown on selection media
![Page 7: Bacterial Transformation RET Summer 2007. Overall Picture Bio-Rad pGLO Transformation Insertion of GFP gene into HB101 E. coli.](https://reader035.fdocuments.in/reader035/viewer/2022072009/56649d8e5503460f94a765ee/html5/thumbnails/7.jpg)
Restriction Enzymes• Endonucleases:
– in nature, they protect bacteria from intruding DNA
– cut up (restrict) the viral DNA
– cut only at very specific nucleotide sequences
• Restriction site:
recognition sequence for a particular restriction enzyme
• Restriction fragments:
segments of DNA cut by
restriction enzymes in a reproducible way
• DNA ligase:
joins the sticky ends of DNA fragments
![Page 8: Bacterial Transformation RET Summer 2007. Overall Picture Bio-Rad pGLO Transformation Insertion of GFP gene into HB101 E. coli.](https://reader035.fdocuments.in/reader035/viewer/2022072009/56649d8e5503460f94a765ee/html5/thumbnails/8.jpg)
Overall Transformation Process
1. The plasmid vector must be cut with a restriction endonuclease (aka: restriction enzyme)
2. DNA ligase joins the DNA fragment & vector DNA
3. Host cell is made competent so can plasmid can enter
4. Transformed cells are grown on selection media
![Page 9: Bacterial Transformation RET Summer 2007. Overall Picture Bio-Rad pGLO Transformation Insertion of GFP gene into HB101 E. coli.](https://reader035.fdocuments.in/reader035/viewer/2022072009/56649d8e5503460f94a765ee/html5/thumbnails/9.jpg)
Transformation of Bacteria
• Generally occurs through heat shock and addition of a divalent cation to permeabilize the membrane
• Competent cells are those capable of taking up the plasmid
![Page 10: Bacterial Transformation RET Summer 2007. Overall Picture Bio-Rad pGLO Transformation Insertion of GFP gene into HB101 E. coli.](https://reader035.fdocuments.in/reader035/viewer/2022072009/56649d8e5503460f94a765ee/html5/thumbnails/10.jpg)
Overall Transformation Process
1. The plasmid vector must be cut with a restriction endonuclease (aka: restriction enzyme)
2. DNA ligase joins the DNA fragment & vector DNA
3. Host cell is made competent so can plasmid can enter
4. Transformed cells are grown on selection media
![Page 11: Bacterial Transformation RET Summer 2007. Overall Picture Bio-Rad pGLO Transformation Insertion of GFP gene into HB101 E. coli.](https://reader035.fdocuments.in/reader035/viewer/2022072009/56649d8e5503460f94a765ee/html5/thumbnails/11.jpg)
Selection• A selective medium is used to determine which
bacterial cells contain the antibiotic resistant plasmid insert and which do not
• For example, a bacterium containing a plasmid with resistance to a particular antibiotic (ampicillin) will grow on medium that contains that antibiotic
• In addition, our plasmid contains a regulatory element that activates the GFP gene only in the presence of arabinose
![Page 12: Bacterial Transformation RET Summer 2007. Overall Picture Bio-Rad pGLO Transformation Insertion of GFP gene into HB101 E. coli.](https://reader035.fdocuments.in/reader035/viewer/2022072009/56649d8e5503460f94a765ee/html5/thumbnails/12.jpg)
Selection Media
LB plates:
LB + amp:
LB + amp + ara:
Control (-pGLO)
Should contain only cells with the amp-resistant pGLO plasmid; colonies appear white (-pGLO, + pGLO)
Should contain only cells with the amp-resistant pGLO plasmid; colonies floresce green (+pGLO)
![Page 13: Bacterial Transformation RET Summer 2007. Overall Picture Bio-Rad pGLO Transformation Insertion of GFP gene into HB101 E. coli.](https://reader035.fdocuments.in/reader035/viewer/2022072009/56649d8e5503460f94a765ee/html5/thumbnails/13.jpg)
Factors that Affect Yield and Quality of Plasmid DNA
• Plasmid copy number
• Host strain used, carbohydrate production
• Culture medium, selection, and culture time– Want to harvest during log growth phase
![Page 14: Bacterial Transformation RET Summer 2007. Overall Picture Bio-Rad pGLO Transformation Insertion of GFP gene into HB101 E. coli.](https://reader035.fdocuments.in/reader035/viewer/2022072009/56649d8e5503460f94a765ee/html5/thumbnails/14.jpg)
Transformation Applications
![Page 15: Bacterial Transformation RET Summer 2007. Overall Picture Bio-Rad pGLO Transformation Insertion of GFP gene into HB101 E. coli.](https://reader035.fdocuments.in/reader035/viewer/2022072009/56649d8e5503460f94a765ee/html5/thumbnails/15.jpg)
GFP Uses• Use as a reporter molecule to
follow changes in gene expression over time
• Nondestructive, nontoxic
• Coding sequence can be cloned into a variety of vectors
• GFP keeps its fluorescence in cells from different species
• Can be tracked in living cells over to time to study development
• Can be directed to specific subcellular compartments
• Can combine GFP coding region with the regulatory region for another gene and observe changes in gene expression
• Can be used to make a fusion protein to study localization, turnover & intracellular associations of native protein
• GFP gene is switched on when cells are grown in the presence of arabinose