Lab 6: Transformation
What will our transformations result in?
10^3-10^4 transformed cells per micro diagram of plasmid DNA
What will commercially produced cells transform?
10^7-10^8 colony forming units per microgram of DNA or cfu/ug
What will electroporation result in?
10^9-10^10 transformants per ug plasmid DNA
What is Green fluorescent protein (GFP)?
Aequorea victoria jellyfish gene
Where can E.coli's most important genes be found?
All of the genes required for basic survival and reproduction are found in the single chromosome
In nature, what are bacteria able to do?
Alter their genetic makeup and acquire new characteristics
What is beta lactamase (bla)?
Ampicillin Resistance
Why would you expect to NOT see growth of the control cells on the LB Agar containing ampicillin?
Ampicillin is an antibiotic
What are some examples of transformation events?
Antibiotic resistance, the ability to produce toxins, etc.
What activates the EGFP gene on the plasmid to be used in the bacterial cells?
Arabinose sugar
What are examples of genetic engineering?
Bacteria that produces human insulin, Golden rice, insecticide corn, and glow-in-the-dark mice
What does the Amp gene allow?
Bacterium to make beta-lactamase enzyme
What is a repressor?
Binds to operator to turn off gene expression
What is an oncogene?
Cancer causing genes
What are competent cells?
Cells that are able to take up exogenous deoxyribonucleic acid (DNA) or genetic material
What ir ORI needed for?
Cells to make copies of the plasmid DNA
If only 1 in every 10,000 cells is successfully transformed, why do you think other people in the class have different numbers of colonies?
Cells were not exactly the same
What are tumor suppressor genes?
Changes in genes that prevent uncontrolled cell growth
What is the central dogma?
DNA to RNA to Protein to Trait
Does E. coli have a nucleus?
Do not have a nuclear envelope surrounding the chromosome and thus, no true nucleus
What is another method of transformation?
Electroporation
How does beta lactamase secrete?
Extracellularly
What does araC regulate?
GFP transcription
What is a regulator?
Gene that encodes for a repressor protein
What does the beta-lactamase do?
Inactivates ampicillin
What can make cells more competent or "pick up" more DNA (2)?
Incubating cells on ice with divalent cations (Ca2+) and with naked DNA
What is electroporation?
Involves incubating plasma DNA wit cells and exposing them to an extremely high electrical field
What is an inducer?
Joins with an active repressor and inactivates it
What is a co-repressor?
Joins with an inactive repressor and activates it
What 3 features in this protocol make the E.coli cells more competent to "pick up" the plasmid DNA?
LB Broth, Temperature shock, and CaCl2 solution
What is the operator region?
Like a traffic cop
What are plasmids?
Loops of DNA found in some bacteria
What is the process of genetic engineering?
Manually adding new DNA to an organism
What is E.coli?
Most common bacterium in the human gut
What should you expect to see growth of the control cells on the lB Agar?
No antibiotics on plate
What is a proto-oncogene?
Normal cellular genes
What does an operator site control?
Several genes
What does pGLO contain?
Short DNA sequences known as the Origin of replication, or ORI
What does plasmid contain?
Special genes that are not related to basic life functions of bacteria
What is transformation?
The ability for bacteria to alter their genetic makeup and acquire new characteristics
What difference, if any, did you see for the colonies growing on the Ampicillin plates that contained arabinose sugar when you exposed them to the UV light?
They glow under UV light
Why would you need a smaller volume of commercially prepared cells?
To get the equivalent number of colonies on a plate as a result of the transformation procedure since they are more efficient
True of false: plasmids can be transferred from one bacteria to another.
True
What is another definition for transformation?
Uptake of naked DNA in the environment by a recipient bacterium
What happens if the plasmid does not have an ORI?
Will not be copied and is not passed down to the daughter cells
Why put the experimental cells with plasmid DNA onto media with Ampicillin?
You want to see the ones that would survive the Ampicllin
What is araC?
araC regulator protein
What is an operator?
"on/off" switch for operon
What is an operon?
Group of genes expressed simultaneously
What is the formal definition of an operon?
Group of structural genes regulated as a unit
What can make cells more competent or "pick up" more DNA (1)?
Growing cells to mid-log phase (typically around 3 hours after inoculation)
What can make cells more competent or "pick up" more DNA (3)?
Heat shocking the cells and DNA at 42C
What does a successful transformation of bacteria require?
High concentration of cells in solution, and only a fraction of the cells that are competent will undergo transformation
What happens during electroporation?
Plasma DNA will migrate towards positive charge in this electrical field, and penetrate the cell wall and membrane of the cells in the solution
What is a pGLO?
Plasmid that carries antibiotic resistance gene that confers resistance to Ampicillin
How fast can E. coli reproduce?
Very rapidly; a single microscopic cell can divide to form a visible colony with millions of cells overnight