General Biology II - Lab Exam
Terms
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- The sequences on MCS recognize _____.
- The sequences on MCS recognize many different RESTRICTION ENZYMES.
- The beta-galactosidase gene is also called _____.
- lacZ gene
- Plasmids can be designed to make copies of _____.
-
- DNA
- mRNA
- protein - procedure for discarding recombinant e. coli cells
-
- liquids: bacterial waste flask
- solids: biohazard bags - Ca++ ions
- Required for CLASSICAL PATHWAY to function
- The MCS serves as the site for _____.
- The MCS serves as the site for DNA INSERTION (and is opened by restriction enzymes).
- The MCS occurs _____.
- The MCS occurs in the middle of the lacZ gene.
- Haemophilus influenzae & Hind II
- The first restriction endonuclease enzyme, called Hind II, was isolated from Haemophilus influenzae. It recognized only one specific 6 bp dsDNA sequence.
- parameters that effect how fast molecules move through the gel during electrophoresis
-
- agarose concentration
- voltage applied to gel
- shape of the DNA (linear, circular)
- molecular size of DNA fragment - Mg++
- Required for ALTERNATIVE PATHWAY to function
- competence factors
-
1. The phase of the bacterial growth cycle at the time of collection"
- competence develops as culture ceases exponential growth
- competency common in eary stationary phase, but only small portion actually becomes competent
2. The media in which the cells are grown:
- nutritional conditions may be a limiting factor, causing competence - vector
- DNA molecule designed to carry a cloned DNA fragment
-
Using Bacterial Plasmids to Clone Genes
Put the following events in chronological order:
- transformation
- use of enzyme ligase
- use of restriction endonuclease
- reproduction of bacteria -
1. RESTRICTION ENDONUCLEASES are used to cut the plasmid and isolate the DNA source.
2. ENZYME LIGASE is used to incubate the fragments together in a ligation reaction.
3. The products of the ligation reaction are TRANSFORMED into competent bacteria cells.
4. The transformed bacteria REPRODUCE. - three commonly used vectors
-
- plasmids
- phages
- artificial chromosomes - ligation reaction
- Many copies of the cut plasmid fragments and cut source DNA fragment are incubated together with the enzyme LIGASE.
- DNA standard curve x-axis
- DISTANCE migrated from well
- erythrocytes
- red blood cells
- stop buffer
-
- added to mixture after incubation of 30 minutes
- phosphate buffered saline with EDTA that chelates cations such as Ca++ and Mg++ so they are not available - Easiest and most commonly used vector type.
- plasmids
- Ligase forms _____ bonds between the ends of the plasmid and DNA insert.
- Ligase forms PHOSPHODIESTER bonds between the ends of the plasmid and DNA insert.
-
TRUE/FALSE
Enzymes that recognize the MCS are usually very specific and will not cut the plasmid at other sites. - TRUE
- recognition sequence
- Also restriction site, it is the sequence on the DNA that is specific for its matching restriction endonuclease enzyme.
- DNA standard curve y-axis
- Log base 10 of DNA fragment SIZE
- white blood cells
-
- neutrophils
- lymphocytes
- monocytes
- eosinophils
- basophils - histones
-
- exclusively prokaryotic
- proteins found on chromosomes - conjugation
- direct cell-cell contact and DNA exchange via pili formation
- Disadvantage of using plasmids as cloning vector.
- DNA fragment must be relatively small (<15 kb)
- After the ligation reaction, the products are transformed into _____ bacterial cells.
- The products transform into COMPETENT cells.
- lacZ gene
-
- part of the lac operon
- produces enzyme beta-galactosidase - methylation of DNA
- Methylation of DNA prevents the restriction enzymes from cutting the recognition sequence on the enzyme's own sequence on the bacterial DNA.
- P-20
- The name of the micropipettor used that holds up to 20 MICROLITERS.
- components found in a blood sample
-
- RBCs
- WBCs
- plasma
- platelets - organelles exclusive to eukaryotes
-
- Golgi apparatus
- mitochondria
- chloroplasts
- endoplasmic reticulum
- nuclear envelope - transformation
-
- ability to take in an incorporate exogenous DNA into the genome
- naturally occuring in few species, must be induced in E. coli - transformation of B. subtilis
-
- begins with reversible binding of donor DNA to surface of competent cells
- DNA is then irreversibly bound to cell and becomes insensitive to DNase which normally destroys DNA
- one strand of donor dsDNA is taken in and one is degraded
- remaining strand is integrated into homologous region of main chromosome - Plasmids replicate [independently] or [dependently] from the main bacterial chromosome.
- independently
- Competent cells can take up _____.
- Competent cells can take up EXOGENOUS DNA from their surroundings such as plasmid DNA.
- What is the effect of combining X-gal and beta-galactosidase?
- X-gal causes beta-galactosidase to produce a blue dye making the bacteria look blue.
- ethidium bromide
- An intercalating dye that, when present, causes DNA to flouresce a bright orange-pink color.
- MBL
-
Mannan-Binding Lectin Pathway:
Initiates complement activation after MBL binds to certain encapsulated bacteria. - blood serum
-
- plasma minus the clotting factors
- still contains antibbodies, proteins, sugars, etc. - organelles in prokaryotes
-
- ribosomes (also in eukaryotes)
- cell walls
- capsules
- inclusion bodies
- gas vacuoles
- flagella - naked DNA
- DNA with no surrounding cell structures
- site specific recombination
- recombination that requires integration into homologous regions
- ______ binds to the plasmid's origin for DNA replication (ori).
- DNA polymerase
-
P+ = Reproduction rate of bacteria containing plasmids.
P- = Reproduction rate of bacteria containing no plasmids.
Relate the two variables:
P+ ___ P- (< , > , =) -
P+ < P-
Bacteria with plasmids take longer to generate because plasmid DNA requires the same "building block" molecules as the main chromosome. - In the gel electrophoresis lab, what is the difference between a blue bacterial colony and a white colony?
-
BLUE contains:
- plasmid
- NO DNA insert
WHITE contains:
- plasmid
- DNA insert (correct/maybe incorrect) - Southern transfer
- DNA fragments in the agarose gel are transferred to a nitrocellulose membrane for permanent storage.
- Alternative Pathway
-
- activates the complement cascade
- does not require presence of antibody
- pathogen's surface molecules initiate activation directly - method of extracting serum
-
- collect blood without an anticoagulant
- let it sit until it clots
- centrifuge the sample
- the liquid is serum - operon
- a set of genes with related functions that are transcribed as one mRNA from one promoter
- Plasmids often carry genes for resistance to __________.
- antibiotics
-
Plasmid DNA is added to an agar plate containing a strain of non-competent E. coli cells.
Will transformation occur? Why or why not? - NO. Only competent cells can take in exogenous DNA such as the plasmid DNA in this example. Therefore, transformation will not occur.
- In the gel electrophoresis lab, you've separated the blue colonies from the white colonies. Do all the white colonies contain a DNA insert?
- YES. The white colonies carry a DNA insert, although not necessarily the correct one.
- Northern transfer
- RNA fragments are separated by gel electrophoresis then transferred from the gel to a membrane for storage.
- Classical Pathway
-
- activates complement system
- initiated b y binding of antibody to antigen on pathogen cell surface
- requires prior specific immunity to create the specific antibody needed - visual sign of RBC lysis
- the normally clear, golden plasma or serum becomes a pink or reddish color due to the presence of free hemoglobin
- binary fission
- bacterial reproduction mechanism
-
TRUE/FALSE
Most of the plasmids used for cloning produce very few copies of the plasmid per cell. -
FALSE
Most of the plasmids used for cloning produce MANY (tens or hundreds) of copies of the plasmid per cell. - pGEM
- plasmid carrying a gene for ampicillin resistance
- Why don't the white colonies produce a blue dye even when X-gal is added?
- The blue dye requires both beta-galactosidase AND X-gal, and beta-galactosidase isn't transcribed in the white colonies.
- visibility of rRNA, tRNA, & mRNA on agarose gel
-
- rRNA molecules can be seen
- tRNA and mRNA rarely seen without using probes - MAC
-
membrane attack complex
- perforates target cell membrane
- causes lysis of target cell - Gal-3G
-
- sugar molecule
- surface antigen found on rabbit RBC
- human blood contains antibody vs. it - generation time
- the time required for the population to double in size
- required treatment for transformation of E. coli
- treatment with calcium chloride to make cell membranes more permeable
- competence
- the physiological state of cells when they can physically bind homologous DNA to their surface and take it inside
- auxotrophs
- mutants that grow only on media that contains the essential yet missing molcules
- Name three types of DNA sequences commonly found on plasmids used as cloning vectors.
-
- MCS for enzyme recognition
- gene marker to identify bacterial cells lacking the plasmid
- DNA insert recognition - You are color-blind. You test a certain colony of transformed E. coli and discover that its DNA does not contain the lacZ gene sequence. Predict the color of this bacterial colony after adding X-gal.
- WHITE. The lacZ gene transcribes beta-galactosidase, which reacts with X-gal to produce the blue color.
- MCS stands for _____.
- multiple cloning site
- You have just isolated a certain DNA fragment from a mouse and you'd like to clone it for further study using plasmids. Should you choose plasmids with antibiotic resistance? Why?
- YES. Cloning plasmids usually carry an antibiotic resistance gene. This resistance will allow you to separate the transformed cells from those that haven't successfully transformed.
- expression vector
- plasmids that are engineered to produce a large quantity of one cloned protein
- complement system
-
- approx. 20 blood proteins
- sequentially activated only during infection
- activation forms MAC - sialic acid residue
-
- found on sheep RBC membranes
- sugar molecule
- binds to Factor H in human circulatory system which blocks the alternative pathway - transduction
- bacterial DNA transfer via phages