PGLO transformation

How do you find the amount of DNA spread on the agar plate?

(1) Multiply volume by concentration to get total amount of pGLO DNA in ug (2) Find fraction of DNA that was used by using this equation: Fraction of DNA used=Volume spread on LB/amp plate (ul)/Total sample volume in test tube(ul) (e.g. We spread 100ul of cells containing DNA from a test tube containing a total volume of 510 ul of solution;FOD=0.2) (3) Multiply to the total amoung of pGlo by FOD. This value will be the denominator of your transformation efficiency

What two pieces of information do you need to calculate the efficiency of your transformation?

(1) The total number of green fluorescent colonies growing on your LB/amp/ara plate (2) The total amount of pGLO plasmid DNA in the bacterial cells spread on the LB/amp/ara plate.

Give examples of how genetic transformation is used in everyday life.

Agriculture: genes coding for traits such as frost, pest, or spoilage resistance can be genetically transformed into plants. Bioremediation: bacteria can be genetically transformed with genes enabling them to digest oil spills. Medicine: Diseases caused by a defective genes are beginning to be treated by gene therapy; that is, by genetically transforming a sick person's cells with healthy cop

What is the ON/OFF switch for the Arabinose operon?

Ara C

Presence of arabinose initiates binding of RNA binding to ________________ and thus transcription of the genes __________,________, _________, and_________.

AraC Ara B, Ara A, Ara D, GFP

What two factors must be present in the bacteria's environment for you to see the green color and why?

Arabinose and UV light Arabinose will cause the GFP gene to be transcribed creating the fluorescent protein and the UV light helps us to see this glow.

Interaction between _____ and________ causes a change in the shape of ________ which in turn promotes the binding of _______________ and the three genes araB, A, and D are transcribed.

Arabinose and araC araC RNA polymerase

What enzyme does the 'bla' gene encode for?

B-lactamase

What are the two methods that we used to increase uptake of PGLO plasmid through the cell membrane?

CaCl2 (calcium chloride) Heat shocking

Why did we use CaCl2 in this experiment?

CaCl2 was used to make cells competent They will bind to DNA and make it neutral increasing the likelihood that it will come in contact with a negative bacterial cell.

Descendents of genetically engineered cells are called ______.

Clones

How do you find the total number of green fluorescent colonies growing?

Count glowing cells under UV light

How can you prevent reforming of chopped strands?

Cut with different restriction enzymes

What was the bacterium used in this experiment?

E.coli

What was the restriction enzyme that we used in this experiment?

EcoRI

T/F CaCl2 bound to the bacterial cell increases likelihood of uptake.

False: CaCl2 bound to DNA increases likelihood of uptake by negative bacterial cell.

T/F Recent occurrence of bacterial resistance to antibiotics is due to the transmission of chromosomes

False: due to transmission of PLASMIDS

The EcoRI restriction enzyme cuts between bases _______ and ______ creating" ________ _________"

G, A "sticky ends"

The pGLO plasmid has been genetically engineered to carry the _______________ gene which codes for the green fluorescent protein, GFP, and a gene called _______________ that codes for a protein that gives the bacteria resistance to an antibiotic.

GFP gene 'bla' gene

Following transformation, the bacteria express their newly acquired jellyfish gene and produce the fluorescent protein, which causes them to do what?

Glow a brillant green color under UV light

What was GFP in this experiment?

Green Fluorescent protein

What experiment was the first evidence of transformation?

Griffith

Selection for cells that have been transformed with pGLO DNA is accomplished by seeing ____________ on ampicillin plates.

Growth

What were our hypothesis for this experiment?

If bacteria with +pGLO plasmids that are resistant to ampicillin and have the gene for GFP, colonies will survive and grow on LB/amp +pGLO bacteria on plate with LB/amp/ara will grow and grow green because of inclusion of arabinose. In control plates, -pGLO bacteria that are amp sensitive will not be able to grow on LB/amp plates. The other control plate with -pGlo bacteria and no ampicillin will grow.

What is an LB plate?

Luria broth (LB) is a nutrient-rich media commonly used to culture bacteria in the lab. The addition of agar to LB results in the formation of a gel that bacteria can grow on, as they are unable to digest the agar but can gather nutrition from the LB within.

Would satellite colonies appear when transferred to a medium containing fresh ampicillin?

No, because they will no longer have the bla expressing colonies to provide a favorable environment for them to survive in.

What is the name of the arabinose promoter?

Pbad

For transformed cells to grow in the presence of ampicillin you must do what?

Provide them with nutrients and a short incubation period to begin expressing their newly acquired genes (cloning genes)

___________ ___________ recognize ___________ sequences and chop the ____________ __________backbone.

Restriction enzymes palindromic sugar-phosphate

What are satellite colonies and why did they appear in this experiment?

Satellite colonies are very small colonies of cells that have not taken up the plasmid that form around a large colony that has taken up the bla-containing plasmid. The satellites form because the beta-lactamase released by the bla-expressing colony degrades the ampicillin in the vicinity of the colony.

Provide a description of E.coli colonies on nutrient agar.

Smooth grayish-white 1-3mm entire edge uniformaly circular with smooth edges Shape (form): circular Margin: entire Elevation: raised Size: punctiform, small Texture (surface): smooth Appearance: shiny Pigmentation: nonpigmented (colorless) Optical property: translucent

E.coli without engineered plasmid will be (resistant/susceptible)________________ to ampicillin.

Susceptible

What does the pGLO plasmid look like?

The DNA code of the pGLO plasmid have been engineered to incorporate aspects of the arabinose operon. Both the promoter (Pbad) and the araC gene are present. However, the genes for araB, D, and A are replaced by gene that codes for GFP. Therefore, in the presence of arabinose, araC protein promotes the binding of RNA polymerase and GFP is produced.

What specifically causes the glow in transformed cells?

The expression of the GFP gene into actual green fluorescent protein.

The gene for GFP can be switched on in transformed cells by adding __________ to the cells nutrient agar medium.

The sugar arabinose

Why would bacteria want restriction enzymes?

They are constantly under attack from bacteriophages so with this ability they can recognize DNA that is not their own and chop it.

The goal of genetic transformation is to change an organism's __________ also known as phenotypes.

Traits

___________ ____________ is the quantitative measurement used to determine the extent to which you genetically transform cells.

Transformation Efficiency helps scientists determine how well transformation is working.

What is the formula for transformation efficiency?

Transformation Efficiency= Total # of colonies growing on agar plate/ Amount of DNA spread on the agar plate (in ug)

Two possible sources of fluorescence within the colony can often be falsely concluded to be the DNA or the green fluorescent protein. However, how did we prove that it was not due to the DNA?

When shining UV light on the sample of original pGLO plasmid DNA no glowing occurred so the glowing was solely due to the expressed green fluorescent protein.z

Transformed cells not containing arabinose will appear___________ and ___________ when arabinose is present.

White(wild-type phenotype) Fluorescent green

What is pGLO?

a type of genetically engineered plasmid that encodes the gene for GFP and a gene for resistance to the antibiotic ampicillin. Also incorporates a special gene regulation system that can be used to control expression of the fluorescent protein in transformed cells.

What are competent bacteria?

bacteria that have the ability to take up free, extracellular genetic material from the environment

What is the source of the gene for GFP?

bioluminescent jellyfish Aequorea victoria

What does B-lactamase do?

cleaves B-lactam ring on ampicillin causing it to not be functional anymore.

What is Bio-Rad?

company that offers a range of products and services for life science research and education, clinical diagnostics, biopharmaceutical processing, and food science.

You can generate ____________ ends that match with "sticky ends".

complementary

Scientist use a process called ___________ __________ to insert genes coding for new traits into plasmid.

genetic engineering

What is genetic transformation?

literally means "change caused by genes" and involves the insertion of a gene into an organism in order to change the organism's trait. Transformation is a process of horizontal gene transfer by which some bacteria take up foreign genetic material (naked DNA) from the environment

What do the genes araB, araA, and araD code for?

three digestive enzymes involved in the breakdown of arabinose