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78 Cards in this Set

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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.
Plasmids can be designed to make copies of _____.
- DNA
- mRNA
- protein
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
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
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