question

What is the 1st step of the P1 lytic cycle?

answer

Adhesion of the bacteriophage to E. coli via tail fibers to the E. coli outer membrane (calcium dependant)

question

What type of bacteriophage is P1?

answer

Lytic

question

What is a generalized transducing phage?

answer

P1 randomly packages E. coli chromosome bits accidentally instead of its own genome, and carries this E. coli fragment out of the cell during lysis.

question

Generalized transduction requires ____________?

answer

homologous recombination take place between the accidentally packaged and transferred E. coli gene segment and that of the recipient E. coli genome.

question

How frequently does homologous recombination occur in E. coli?

answer

typically at 0.3% of the bacteriophage will actually have accidentally picked up your desired E. coli chromosomal fragment

question

What must be used to screen for proper transductants?

answer

Antibiotic selectable marker

question

Calcium Chloride (CaCl2)

answer

Is required for the tail fibers of the P1 to be in the correct conformation to allow binding to the LPS of the recipient E. coli.

question

Sodium Citrate (NaCit)

answer

Is required to sequester the remaining calcium ions after initial infection. This prevents further P1 infection and prevents lysis of the properly transduced E. coli.

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Incubation at 37oC for 30 minutes

answer

Is also required to allow expression of the newly recombined antibiotic resistance gene. Without this incubation prior to plating on the selection media, the antibiotic resistance gene would not have time to be expressed and protect the cell from the selection media.

question

What is the 2nd step of the P1 lytic cycle?

answer

Injection of the bacteriophage DNA into the E. coli host cell

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What is the 3rd step of the P1 lytic cycle?

answer

Replication of bacteriophage DNA and synthesis of phage proteins

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What is the 4th step of the P1 lytic cycle?

answer

Maturation of bacteriophage particles to mature P1

question

What is the 5th step of the P1 lytic cycle?

answer

Lysis of the host E. coli cell

question

Part 1 – Alkaline lysis miniprep isolation of the plasmid

answer

Purpose of this step is to isolate a plasmid from one strain of E. coli for transfer to another strain of E. coli.

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Tris-EDTA (TE)

answer

Used as a buffer solution at pH 8.0, where DNA is most stable. sequesters divalent cations to prevent the activity of DNases, which require Mg2+ for enzymatic activity.

question

Sodium Hydroxide/Sodium Dodecyl Sulfate (NaOH–SDS)

answer

solubilizes membranes and denatures proteins. Once the cell membrane has been dissolved and the proteins denatured, the cell lyses and the cellular contents are released. Denatures DNA from double-stranded to single-stranded.

question

Potassium Acetate (KAc)

answer

Brings the pH back to neutral. This allows the smaller DNA molecules (the plasmids) to return to the double-stranded conformation, but keeps the large chromosomal DNA denatured. Interacts with denatured proteins, denatured DNA and solubilized membranes, causing them all to precipitate out of the solution. Only the plasmid DNA remains in the supernatant.

question

95% Ethanol

answer

Forms hydrogen bonds with water, disrupting the DNA from forming hydrogen bonds with the water. As a result, the plasmid DNA precipitates in the solution.

question

70% Ethanol

answer

Forms hydrogen bonds with water, disrupting the DNA from forming hydrogen bonds with the water. As a result, the plasmid DNA precipitates in the solution.

question

Part 2 – Agarose gel electrophoresis

answer

Gel electrophoresis was performed in this experiment to determine if you isolated DNA, as well as to quantify the amount of DNA isolated.

question

What is the gel matrix made from?

answer

0.8% agarose (super purified agar) is used to separate DNA molecules based on size (length in base pairs) under an electric field.

question

Gel electrophoresis - Larger DNA molecules will __________?

answer

run slower (stay closer to the negative electrode) because it is harder for them to squeeze through the matrix.

question

Gel electrophoresis - Smaller DNA molecules will __________?

answer

move faster (closer to the positive electrode) because they can weave easier through the gel.

question

Gel elctrophoresis - DNA runs towards what ________?

answer

cathode (positive - red) because of a net negative charge on the molecule conferred by the negatively charged phosphodiester backbone.

question

Ethidium Bromide

answer

was used to visualize the DNA in the gel under UV transillumination

question

intercalating agent

answer

binds to the bases of DNA and fluoresces orange under UV light

question

The amount of fluorescence can be used to determine ?

answer

the amount of DNA present in the sample.

question

What's the purpose of Calcium Transformation?

answer

introduce the isolated plasmid into a recipient E. coli strain. To do this, the cell membrane was treated with Mg2+/CaCl buffer and then subjected to a heat shock of varying times at 42oC.

question

Calcium Buffer

answer

Chemically alters the E. coli in order to allow the membranes to permit passage of plasmid DNA into the cell.

question

Calcium buffer contains ________ ?

answer

magnesium and calcium ions in high concentrations, with calcium being the most important.

question

Calcium Buffer's mechanism?

answer

which this buffer makes E. coli competent to take up DNA is not understood.

question

Heat Shock (42oC)

answer

Done to the competent cells to increase the fluidity of the bacteria’s cell membranes in order to allow the plasmid DNA to pass through.

question

The length of the heat shock should ________?

answer

in theory, correlate with the efficiency of plasmid being taken up by the bacteria. Too short, and the membrane would not be fluid enough, too long, and the cell could die.

question

Post Heat Shock Cooling (2 minutes on ice)

answer

This step allows the bacteria to recover from the heat shock. The bacteria can repair their membranes before starting a growth cycle in LB broth. The salt in LB broth (0.5%) is too high for bacteria at this time, and would kill them if they have not repaired their membranes.

question

Incubation at 37oC for 30 minutes

answer

Similar to the P1 transduction step. Gives time for the E. coli to express any new genes (including the ones for antibiotic resistance) that have been acquired on the plasmid, allowing for selection on antibiotic media.

question

% survivors

answer

tells the researcher what per cent of the original cells survived the heat shock.

question

% transformed

answer

tells the researcher what per cent of the cells survived heat shock and took up a plasmid.

question

Transformation Frequency

answer

allows the researcher to compare the results from one experiment to the next.

question

transformation frequency takes into account

answer

how much DNA was added to each aliquot (usually 100 ?l) of competent cells. The likelihood that a bacterium will take up a plasmid is dependent not only on whether the bacterial membrane allows passage of the plasmid into the cell, but also how much DNA is present outside the cell to be taken up.

question

Protein Fractionation and Enzyme Analysis Experiment - What was the purpose?

answer

begin the process of isolating one particular protein (?-galactosidase) away from the rest of the 2000+ proteins present in the E. coli cell.

question

Cell lysis

answer

Lysis of the E. coli cells was performed using a Sonicator

question

What is a Sonicator?

answer

sonicator submits the bacteria to high frequency sounds waves that disrupt the membrane and lyse the cells. These high frequency sound waves also act to shatter all DNA in the cell (chromosomal and plasmid).

question

MOPS

answer

Standard buffer that is at pH 7.5, allowing for stability of proteins.

question

Dithiothreitol (DTT)

answer

Creates a reducing environment. The cytoplasm of E. coli is also a reducing environment. Therefore, to keep proteins stable, we recreated this environment in the buffer.

question

Benzamide

answer

Inhibits proteases from degrading the proteins from the lysed cells.

question

Lysate Fractionation

answer

involves the “salting out” of proteins. This refers to the disruption of the protein’s hydrogen bonding with water.

question

What was used as the salt disrupter in lysate fractionation?

answer

Ammonium sulfate (NH3SO4) was used

question

Ammonium sulfate (NH3SO4) does what?

answer

bonding with the water in place of the proteins. Lack of hydrogen bonds with water causes proteins to precipitate in the solution.

question

The Crude Extract

answer

No ammonium sulfate added. Represents all of the proteins in the cell.

question

0-40% Fraction

answer

Concentration of ammonium sulfate increased to fourty%. Represents all proteins whose hydrogen bonding with water was disrupted by this addition of the salt.

question

40-80% Fraction

answer

Concentration of ammonium sulfate increased to eighty%. Represents all proteins whose hydrogen bonding with water was disrupted by this addition of the salt.

question

>80% Fraction

answer

Represents the proteins that require more than 80% ammonium sulfate to break their hydrogen bonds with water and be precipitated.

question

Bradford

answer

colorimetric method employed in class A595

question

Coomassie Blue G

answer

Detect basic amino acids as well as aromatic amino acids.

question

Coomassie Blue G - Advantages

answer

include high sensitivity, very fast, and little interference.

question

Coomassie Blue G - Disadvantages

answer

the specificity of the amino acids detected, differing concentrations of those amino acids between your sample and the standard used.

question

Beta galactosidase

answer

enzyme produced by the lacZ gene that cleaves the disaccharide lactose into the monsaccharides glucose and galactose

question

Enzyme activity

answer

refers to the amount of beta-galactosidase in solution and it is quantified in terms of how much substrate it converts to product in a dfined period of time (usually 1 hour)

question

ONPG

answer

cleaved by beta-galactosidase and the cleaved products create a yellow color that we can measure at an absorbance of 420 nm

question

Cellular debris

answer

absorbance reading of 550 nm which will tell you the concentration of the debris

question

Specific activity

answer

used in order to be able to compare the different protein fractions in terms of how much beta-galactosidase is present with respect to other proteins.

question

Best fraction contained _______?

answer

the most beta-galactasidase while containing the least amount of of other proteins.

question

Bradford Assay

answer

amount of protein in each fraction

question

Bradford Assay was compared to ?

answer

BSA standard protein curve

question

Units/mg of protein

answer

The enzyme units of each fraction were then divided by the concentration of protein in each fraction

question

The specific activity is what was used __________

answer

to determine which fraction gave the best yield of beta-galactosidase

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