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