Download - Bacterial Transformation
![Page 1: Bacterial Transformation](https://reader035.vdocuments.net/reader035/viewer/2022062423/56814565550346895db237bd/html5/thumbnails/1.jpg)
Bacterial Transformation
![Page 2: Bacterial Transformation](https://reader035.vdocuments.net/reader035/viewer/2022062423/56814565550346895db237bd/html5/thumbnails/2.jpg)
What is transformation?
Changing the genes and phenotype of a bacteria by uptake of foreign/new DNA
Let’s review bacterial DNA first…
![Page 3: Bacterial Transformation](https://reader035.vdocuments.net/reader035/viewer/2022062423/56814565550346895db237bd/html5/thumbnails/3.jpg)
Bacterial genome Bacteria are
prokaryotes—no nucleus. The area where
DNA is located is called the nucleoid
DNA is organized in one double stranded circular molecule
![Page 4: Bacterial Transformation](https://reader035.vdocuments.net/reader035/viewer/2022062423/56814565550346895db237bd/html5/thumbnails/4.jpg)
![Page 5: Bacterial Transformation](https://reader035.vdocuments.net/reader035/viewer/2022062423/56814565550346895db237bd/html5/thumbnails/5.jpg)
What is carried on the Plasmid?
The plasmid contains genes necessary for survival and can be passed from one bacteria to another
Antibiotic Resistance: Some bacteria have genes coding for enzymes that destroy certain antibiotics!
![Page 6: Bacterial Transformation](https://reader035.vdocuments.net/reader035/viewer/2022062423/56814565550346895db237bd/html5/thumbnails/6.jpg)
The transformation lab… Our plasmid—pBlu
plasmid
Into E. coli (scary?…no!)
Our plasmid contains genes for: AMP= ampicillin (an
antibiotic) resistance Beta-galactosidase-an
enzyme that converts X-Gal Indo Blu
Protein that allows for antibiotic resistance
Enzyme that breaks down X-Gal to make Indo Blu
RNA
RNA
![Page 7: Bacterial Transformation](https://reader035.vdocuments.net/reader035/viewer/2022062423/56814565550346895db237bd/html5/thumbnails/7.jpg)
![Page 8: Bacterial Transformation](https://reader035.vdocuments.net/reader035/viewer/2022062423/56814565550346895db237bd/html5/thumbnails/8.jpg)
How do we get the plasmid inside of the bacteria?
1. Obtain E. Coli bacteria cells + Add to CaCl2 (helps plasmid attach to bacteria)
2. Add plasmid to same microtube
1. E. Coli2. pBlu plasmid
![Page 9: Bacterial Transformation](https://reader035.vdocuments.net/reader035/viewer/2022062423/56814565550346895db237bd/html5/thumbnails/9.jpg)
![Page 10: Bacterial Transformation](https://reader035.vdocuments.net/reader035/viewer/2022062423/56814565550346895db237bd/html5/thumbnails/10.jpg)
How do we get the plasmid inside the bacteria?
Wait…and then
3. Heat shock! This temporarily opens pores to allow the plasmid to enter the bacteria…timing is critical!!!
![Page 11: Bacterial Transformation](https://reader035.vdocuments.net/reader035/viewer/2022062423/56814565550346895db237bd/html5/thumbnails/11.jpg)
Growing the bacteria
After they have received the plasmid…
Placed on a growth media and allowed to grow.
![Page 12: Bacterial Transformation](https://reader035.vdocuments.net/reader035/viewer/2022062423/56814565550346895db237bd/html5/thumbnails/12.jpg)
How will we know if the bacteria actually got the plasmid??
Any ideas?
We can grow the bacteria on a plate: That contains ampicillin and X-Gal Regular bacterial medium
What do you predict will happen in each?
![Page 13: Bacterial Transformation](https://reader035.vdocuments.net/reader035/viewer/2022062423/56814565550346895db237bd/html5/thumbnails/13.jpg)
Predict
pBlu
pBlu
Control
Control
Amp
X-Gal
Regular
Amp
X-Gal
Regular
What will we observe???
![Page 14: Bacterial Transformation](https://reader035.vdocuments.net/reader035/viewer/2022062423/56814565550346895db237bd/html5/thumbnails/14.jpg)