BIOtech

What 3 gene are on the Puc18 plasmid.

-ampr "ampacillin resistance allows for growth on xgal media -lacz "codes for b-galactosidase to give off blue color origin of reap- where replication starts

Describe two ways a student can prove his bateria has the PUC18 plasmid.

1st way is if there was growth when the ecoli was cultured xgal media. That means there is a plasmid because there is growth. 2nd way is if some colonies are white because that means that transfer of the sheep DNA from PUC18 vector was inserted into the Cell, so the cell contains plasmid.

How can a scientist turn on/off a gene in a plasmid.

A gene can be turned on /off in a plasmid by gene switches. A repressor can bind to the operator to turn off the gene expression and induser can also bind on to the repressor changes its shape and remove it from the operator to turn on gene expression.

What is a polylinker? how is it used in the lab? Think about restriction endonuclease in the lab.

A polylinker is a small region of DNA that contains many restriction sites. Allowing for the insertion of foreign DNA in the S11 lab.The sheep DNA was inserted in the polylinker that was located on the lac2 gene. The endonuclease restriciton enzyme used in the lab was ECORi1

why do we heat shock and add CaCl?

A rapid rise in temp, or " heat shock" then creates a temp imbalance on either side of the bacterial membrane, and sets up current. With the ionic shield in place the DNA can then be swept through the adhesion zone.

Why do researchers use selectable marker in transformation labs?

A selectable marker allows the experimenter to identify bacterial cells that have taken up the plasmid during the transformation process.

A selectable marker is carried by the vector to..............

A selectable marker is carried by the vector to allow the selection of positively transformed cells. Antibiotic resistance is often used as marker, an example being the beta-lactamase gene, which confers resistance to the penicillin group of beta-lactam antibiotics like ampicillin.

When lactose is present

A small amount of sugar is formed within the bacterial cell. This fits onto the repressor protien at another active site. This causes the repressor protien to change its shape. It can no longer sit on the operator site.RNA polymerase can now reach its promoter site.

The plasmid know as Puc18 ampr contains the DNA base sequence that can be transcribed and translated into a protien enzyme known as beta lactmase. Explain how about bacteria cells picks up ampr plasmid that can grow on lb+ ampicillin

Bacteria cells pick up the ampr plasmid by giving them a heat shock. This heat shock encourages them to pick up the plasmid.

When LacZ is a functional enzyme, X gal is the product. This produces.........

Blue colonies....... researcher is sad

Explain how CACl2 was used in the transformation of the PUC18 say the charges.

CaCl2 was used in the transformation , because there would be bean no growth if CaCL2 wasn't used , because CA+2 creates a positive charged membrane by binding it , it will be attracted to the DNA/PLASMID and take the plasmid in. Once the cell has the plasmid , it can grow and transform on xgal media.

Heat Shock Treatment

Cell uptakes exogenous DNA

Exogenous DNA

DNA outside a cell or an organism

Lactose present

Lactose taken up by bacterium Lactose binds to repressor protien , changes shape and prevents from binding to the operator region on DNA (closes DNA binding site) Transcription no longer inhabited. MRNA produced from three structural gens. This makes sure that the bacterial can produce B-Lactosodise and other things when loctose is available in sourranding medium.

What three components do plasmids contain?

Plasmids contain three components: an origin of replication, a polylinker to clone the gene of interest (called multiple cloning site where the restriction enzymes cleave), and an antibiotic resistance gene (selectable marker).

Constitutive genes

Products made constantly by the cell, synthesis is not regulated. 60-80% of genes

regulated genes

Products made only when needed by the cell. Synthesis is tightly regulated. 20-40% of genes.

When glucose and lactose are present...........

RNA polymerase can sit on the promoter site but it is unstable and keeps falling off.

Lactose absent

Regulator gens codes for repressor protien repressor binds operator region, close to lacz RNA polymerase cant bind two DNA at promoter region. No transcription of the three structural gens

represor protein

Represor protien is continuosly made. It sits of the sequence of DNA just in front of the lac operon, the operator site.

Expalin why researchers use selectable marker........What marker was used in our lab?

Researchers use selectable markers in transformation labs because cells can only be transformed and grow if they have the selectable marker. A selectable marker is a gene introduced into the cells, such as bacteria, that allows for transformation. In the S11 lab the marker is the Ampicillan Resistance bc without it there would be no growth or no transformation

How can we prove in the S11 lab we had ECOLI cells with the plasmid? What happens to these cells if there is no plasmid in the Ecoli on the medium used in the lab?

The Ecoli cells had a plasmid in them bc there was groth/transformation on the plate. If there was no plasmid the cells would be unable to grow.

Why was the betalactamase gene included in the plasmid?

The beta-lactamase gene is included in this plasmid in order to be able to easily test for its presence in bacteria by determining whether they can grow on media containing ampicillan.

Explain clever technique that is used in order to get colonies to turn blue.

The colonies are blue because there was no successfull ligation of the sheep DNA, the lact2 gene was still active. This codes for the b-galactosidase protien , that gives colony of the blue color.

Discuss your understanding of bacterial plasmids as vectors for a gene of interest.

The plasmid from a bacterial cells can be used as Vector by inserting a gene of interest into it. Then transfer the plasmid into a bacterial cells to have the plasmid/vector clone transfer.

What enzyme is xgal broken down by?

When x gal is broken down by beta-galactosidase, it forms a blue color.

Explain parts of the S11 labs.

You can get the gene of interest, ligate it to the plasmid and then the mutation can be curied. Because a new sequence is added in the foreign DNA which causes promoter and operator to hit the sheep DNA instead of the lac operon and it wont recognize this DNA , so the protien wont be coded for.

How can a research make sure they have the gene of interest for cloning from the bacterial colonies.

You can make sure by having the bacteria grow on a media that would have them code for the protien. If the protien is not created like (b-glactasodise) , there will be no effect resulting in white colonies, which means the gene (lac2) was inactivated by the inserted DNA/gene .

How can researchers make sure they have the gene of interest from cloning from their bacterial colonies?

You can make sure by having the bacteria grow on media that would have them code for the protien. If protien is not created like (B- galactosidase), there will be no effect resulting in white colonies which means the gene (lacK z ) was inactvated by the inserted DNA gene

If a bacterial cell contains a plasmid carrying this gene (is a protein/enzyme which can break down pencillan and certain modified pencillans such as ampicillin) then the bacteria can grow in the presence of.................

ampicillan, and are said to have "ampicillan resistant" phenotype

Plasmid mini chromosme code for protiens that.....

are not essential for the survival of their host in its normal enviornment

Plasmid mini chromosome replicates independently of the..........

bacterial chromosome

Lac-Z gene

beta-galactosidase is an enzyme which hydrolizes lactose into component sugars. The activity is measured with chromogenic substances which, when hydrolyzed, produced a colored product. One such substrate is X gal which is converted into a blue product by beta-galactosidase. Bacterial colonies containing the active galactisodase enzyme will appear in blue color.

Repressor protien

blocks the promoter site where the RNA polymerase settles before it starts transcribing

non-recombinant have functional enzyme, their colonies turn..........

blue

Ice cold CACL2 treatment

cell becomes competent

Lacz

coding for b-glactosadise

Beta-lacamase

is a protein/enzyme which can break down pencillan and certain modified pencillans such as ampicillin.

When X Gal is broken down by Beta-galactosidase...........

it forms a blue color. Therefore bacterial cells containing a gene coding for the enzyme will form blue colonies on this agar.

A cell saves energy by only making.......

needed protiens

Theory of Transformation

normal bacterium, CACL2 TREATMENT, after the treatment, plasmid is bound to the exterior of the competent cell, KEPT IN 42 C FOR 2 MINUTES, plasmid has then transported inside of the transformed cell

plasmids can encode protiens that can break down pencillin and certain modified pencillans such as ampicillan

plasmids can encode protiens that can break down pencillin and certain modified pencillans such as ampicillan EXAMPLE; BETA-LACTAMASE

What is puc18? why is it used?

pusc18 is the plasmid from the ECOLI that has ampr and a gene with its promoter for enzyme B galctosidase (lackz). In the S11 lab it was used as a vector/plasmid that contained the inserted sheep dna.

plasmid vector is removed from bacterial cell and cut with a........................

restriction enzyme

Anatomy of a plasmid- mini chromosome

small circular DNA molecule found in bacteria in addition to bacterial chromosome.

plasmids can be designed in the lab to carry........

the gene of interest

Foreign DNA inserted into the LacZ gene destroys the ability of the lacZ gene to be transcribed and....................

translated and an active B-galactosidase protien enzyme is not produced.

pUC18 plasmid

used to help distinguish and screen recombinant clones; observe a blue to white color change in colonies. A common cloning vector

What is a vector?

vector (molecular biology) In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed (e.g., plasmid, cosmid, Lambda phages). A vector containing foreign DNA is termed recombinant DNA.

Recombinant colonies turn........

white

When lacZ is non-functional enzyme, X gal is not formed as the product. This produces.....

white colonies...... researcher is happy