Term
| What is the 1st step of the P1 lytic cycle? |
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Definition
| Adhesion of the bacteriophage to E. coli via tail fibers to the E. coli outer membrane (calcium dependant) |
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Term
| What type of bacteriophage is P1? |
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Definition
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Term
| What is a generalized transducing phage? |
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Definition
| 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. |
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Term
| Generalized transduction requires ____________? |
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Definition
| homologous recombination take place between the accidentally packaged and transferred E. coli gene segment and that of the recipient E. coli genome. |
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Term
| How frequently does homologous recombination occur in E. coli? |
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Definition
| typically at 0.3% of the bacteriophage will actually have accidentally picked up your desired E. coli chromosomal fragment |
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Term
| What must be used to screen for proper transductants? |
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Definition
| Antibiotic selectable marker |
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Term
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Definition
| 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. |
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Term
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Definition
| 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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Term
| Incubation at 37oC for 30 minutes |
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Definition
| 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. |
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Term
| What is the 2nd step of the P1 lytic cycle? |
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Definition
| Injection of the bacteriophage DNA into the E. coli host cell |
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Term
| What is the 3rd step of the P1 lytic cycle? |
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Definition
| Replication of bacteriophage DNA and synthesis of phage proteins |
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Term
| What is the 4th step of the P1 lytic cycle? |
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Definition
| Maturation of bacteriophage particles to mature P1 |
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Term
| What is the 5th step of the P1 lytic cycle? |
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Definition
| Lysis of the host E. coli cell |
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Term
| Part 1 – Alkaline lysis miniprep isolation of the plasmid |
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Definition
| 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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Term
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Definition
| 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. |
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Term
| Sodium Hydroxide/Sodium Dodecyl Sulfate (NaOH–SDS) |
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Definition
| 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. |
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Term
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Definition
| 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. |
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Term
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Definition
| 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. |
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Term
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Definition
| 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. |
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Term
| Part 2 – Agarose gel electrophoresis |
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Definition
| Gel electrophoresis was performed in this experiment to determine if you isolated DNA, as well as to quantify the amount of DNA isolated. |
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Term
| What is the gel matrix made from? |
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Definition
| 0.8% agarose (super purified agar) is used to separate DNA molecules based on size (length in base pairs) under an electric field. |
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Term
| Gel electrophoresis - Larger DNA molecules will __________? |
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Definition
| run slower (stay closer to the negative electrode) because it is harder for them to squeeze through the matrix. |
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Term
| Gel electrophoresis - Smaller DNA molecules will __________? |
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Definition
| move faster (closer to the positive electrode) because they can weave easier through the gel. |
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Term
| Gel elctrophoresis - DNA runs towards what ________? |
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Definition
| cathode (positive - red) because of a net negative charge on the molecule conferred by the negatively charged phosphodiester backbone. |
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Term
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Definition
| was used to visualize the DNA in the gel under UV transillumination |
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Term
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Definition
| binds to the bases of DNA and fluoresces orange under UV light |
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Term
| The amount of fluorescence can be used to determine ? |
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Definition
| the amount of DNA present in the sample. |
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Term
| What's the purpose of Calcium Transformation? |
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Definition
| 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. |
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Term
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Definition
| Chemically alters the E. coli in order to allow the membranes to permit passage of plasmid DNA into the cell. |
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Term
| Calcium buffer contains ________ ? |
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Definition
| magnesium and calcium ions in high concentrations, with calcium being the most important. |
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Term
| Calcium Buffer's mechanism? |
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Definition
| which this buffer makes E. coli competent to take up DNA is not understood. |
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Term
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Definition
| 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. |
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Term
| The length of the heat shock should ________? |
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Definition
| 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. |
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Term
| Post Heat Shock Cooling (2 minutes on ice) |
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Definition
| 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. |
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Term
| Incubation at 37oC for 30 minutes |
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Definition
| 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. |
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Term
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Definition
| tells the researcher what per cent of the original cells survived the heat shock. |
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Term
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Definition
| tells the researcher what per cent of the cells survived heat shock and took up a plasmid. |
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Term
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Definition
| allows the researcher to compare the results from one experiment to the next. |
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Term
| transformation frequency takes into account |
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Definition
| 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. |
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Term
| Protein Fractionation and Enzyme Analysis Experiment - What was the purpose? |
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Definition
| begin the process of isolating one particular protein (-galactosidase) away from the rest of the 2000+ proteins present in the E. coli cell. |
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Term
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Definition
| Lysis of the E. coli cells was performed using a Sonicator |
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Term
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Definition
| 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). |
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Term
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Definition
| Standard buffer that is at pH 7.5, allowing for stability of proteins. |
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Term
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Definition
| 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. |
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Term
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Definition
| Inhibits proteases from degrading the proteins from the lysed cells. |
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Term
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Definition
| involves the “salting out” of proteins. This refers to the disruption of the protein’s hydrogen bonding with water. |
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Term
| What was used as the salt disrupter in lysate fractionation? |
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Definition
| Ammonium sulfate (NH3SO4) was used |
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Term
| Ammonium sulfate (NH3SO4) does what? |
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Definition
| bonding with the water in place of the proteins. Lack of hydrogen bonds with water causes proteins to precipitate in the solution. |
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Term
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Definition
| No ammonium sulfate added. Represents all of the proteins in the cell. |
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Term
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Definition
| Concentration of ammonium sulfate increased to fourty%. Represents all proteins whose hydrogen bonding with water was disrupted by this addition of the salt. |
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Term
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Definition
| Concentration of ammonium sulfate increased to eighty%. Represents all proteins whose hydrogen bonding with water was disrupted by this addition of the salt. |
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Term
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Definition
| Represents the proteins that require more than 80% ammonium sulfate to break their hydrogen bonds with water and be precipitated. |
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Term
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Definition
| colorimetric method employed in class A595 |
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Term
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Definition
| Detect basic amino acids as well as aromatic amino acids. |
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Term
| Coomassie Blue G - Advantages |
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Definition
| include high sensitivity, very fast, and little interference. |
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Term
| Coomassie Blue G - Disadvantages |
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Definition
| the specificity of the amino acids detected, differing concentrations of those amino acids between your sample and the standard used. |
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Term
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Definition
| enzyme produced by the lacZ gene that cleaves the disaccharide lactose into the monsaccharides glucose and galactose |
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Term
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Definition
| 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) |
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Term
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Definition
| cleaved by beta-galactosidase and the cleaved products create a yellow color that we can measure at an absorbance of 420 nm |
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Term
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Definition
| absorbance reading of 550 nm which will tell you the concentration of the debris |
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Term
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Definition
| 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. |
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Term
| Best fraction contained _______? |
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Definition
| the most beta-galactasidase while containing the least amount of of other proteins. |
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Term
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Definition
| amount of protein in each fraction |
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Term
| Bradford Assay was compared to ? |
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Definition
| BSA standard protein curve |
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Term
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Definition
| The enzyme units of each fraction were then divided by the concentration of protein in each fraction |
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Term
| The specific activity is what was used __________ |
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Definition
| to determine which fraction gave the best yield of beta-galactosidase |
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