When you have only one plasmid to isolate, you need a new column for proper balance during
After using a micropipette, it is desirable to set the volume to the maximum.
You use yellow tips for 10 ul micropipettes.
We used RNA primers for PCR amplification.
LB media contains 0.2% glucose.
In Biol 160 it is our normal procedure to take out enzyme and put on ice until use.
For the separation of large DNA fragments, we use a polyacrylamide gel.
For agarose gel electrophoresis, agarose will be dissolved in H2O.
A DNA size marker is composed of different sizes of circular DNA fragments.
Excess use of 6x dye is not recommended because it will affect DNA migration on an agarose gel
We normally do not wash or rinse the flask used for dissolving agarose.
When we made an agarose gel, we added EtBr to the gel.
For plasmid isolation, vigorous mixing is recommended after adding lysis solution
We use the same spin columns for both plasmid isolation and PCR clean-up.
Many bacteria can sense quorum and do so by direct cell-cell communication using pili.
If the genes for luminescence in a bacterial species are under the control of quorum sensing signal, luminescence is dependent upon cell density.
If the genes for luminescence in a bacterial species are under the control of quorum sensing signal, luminescence happens in high cell density above certain concentration.
Bacteria move toward higher concentration of chemical attractants by remembering the previous concentration over time.
Chemoreceptor proteins for chemoeffectors (attractants or repellants) are found in bacterial cell membrane.
The production of the same AI-2 by different types of bacteria in the same community is one way for the bacteria to engage in ( ) communication.
This term is for a community of microorganisms immobilized and living on a solid surface exposed to air or liquid. The obvious advantage would be a formation of food webs via the coordination of different types of microorganisms. What is this?
Chemoreceptor proteins for chemoeffectors are also called ( four words ) (MCPs).
Methyl-Accepting Chemotaxis Protein
( ) are unique among the known prokaryotes in that the cells aggregate and construct multicellular fruiting bodies.
When cultures of B. subtilis undergo nutritional limitation and enter the stationary phase, 10 to 20% of the cells ultimately develop ( ), which allows the uptake of external DNA.
Bacillus subtilis has evolved ways to adjust to stressful changes in the natural environement. One of these ways is to ( a ) when faced with limiting supplies of a carbon or nitrogen source. The decision to ( a ) is regulated by a phophorelay signal transduction system.
The normal flora are always beneficial.
Temperature can be a signal for virulence gene expression for pathogens.
Endotoxins are secreted in soluble form by live bacteria,
An enterotoxin is a protein toxin released by a microorganism in the intestine.
The enzymatic activity of Cholera toxin is ADP-ribosylation.
mRNA can sense environmental signals.
This bacterium, ( ), killed 75-200 million European people (one-third of the population at that time) in 1347 to 1353, known as the Black Death.
This bacterium, ( ) was first isolated by Italian anatomist, Filippo Pacini and also by Robert Koch as the cause of cholera.
This bacterium, ( ) is an opportunistic pathogen, meaning that can cause disease to an immunocompromised person, but not a healthy person. It typically infects lung, so it is a threat especially to a patient having Cystic fibrosis.
( ) species cause gastroenteritis, enteric fever, and septicemia (blood infections)
( ) species cause shigellosis and produces shiga toxin.
This bacterium, ( ) is the causative agent of whooping cough. When you were very young, you probably got an immunization shot against this agent.
This bacterium, ( ) is the most common cause of staph infections, though it may exist as a commensal on human skin.
( ) is the causal agent of Crown Gall disease (the formation of tumors) in over 140 species of flowering plants. The bacterium has a mega-sized Ti plasmid (tumor-inducing plasmid).
When glucose is added to E. coli culture media, you will see a lot of glucose inside the bacterial cell.
In E. coli, glucose the best carbon source so if glucose is around, it represses the utilization of other carbon sources.
In E. coli, cAMP receptor protein is the transcriptional activator involved in catabolite repression. So, the effector (ligand) of the protein is glucose.
When E. coli cells see lactose (a decent carbon/energy source) outside, they always express lactose-utilization enzymes.
RNA polymerase prefers some DNA sites to bind to.
A transcription factor binds to target DNAs by recognizing a specific sequence.
Transcriptional activator helps RNA polymerase transcribe and repressor inhibits RNA polymerase from transcription.
In an operon, the binding site of a transcriptional activator always precedes the site for RNA polymerase (promoter).
In an operon, the binding site of a transcriptional repressor usually precedes the site for RNA polymerase (promoter).
Let’s say you know that the transcription of an operon strictly requires a transcriptional activator. Then the promoter for the operon is strong (& ideal).
How do you call DNA site to which a repressor binds?
Membrane protein cannot be a transcription factor.
Bacteria often use secondary messengers for environmental signals.
Obligatory anaerobes usually have oxygen-sensing transcriptional regulators such as FNR.
To kill host is the best interest for pathogenic bacteria.
Glucose is the best food for E. coli. So, glucose will repress utilization of any other carbohydrate in E. coli.
Many bacteria can sense quorum by direct cell-cell communication using pili.
There is no bacterium that can form multicellular fruiting bodies.
Several bacteria undergo sporulation when faced with nutrient excess.
In two-component systems, the sensor protein is always a transmembrane protein.
When bacteria encounter chemo-attractants (food), they tumble rather than swim.
mRNA can sense environmental signals.
Bacteria sense their environmental changes by recognizing signaling molecules and therefore there must be cellular components to bind to such signaling molecules.
All the environmental signaling molecules go into the cytoplasm in order to trigger appropriate adaptation mechanisms in bacteria.
Upon binding to signaling molecules, transcriptional regulators (or sensor proteins in two-component systems) undergo conformational changes (that is, there must be two protein forms in each case).
The regulation by altering protein’s activity is faster than that by changing the transcription level of the protein.
When glucose is NOT around, several glycolysis enzymes will be down-regulated through the alteration of the proteins’ activities.
The reactions catalyzed by regulatory enzymes are usually at a metabolic branch point.
Regulatory enzymes often catalyze reactions that are physiologically irreversible.
A positive allosteric effector would increase the Km of its protein.
For biosynthetic pathways, the end product is often a negative allosteric effector for a branch point enzyme. Such control is called end-product inhibition, or ( two words ), by an end product.
The ( ) is the substrate concentration yielding 1/2Vmax.
When the effector binds to the ( ) site (the effector site), the protein undergoes a conformational change, and this changes its kinetic constants.
Regulatory enzymes typically show ( ) kinetics, while non-regulated enzymes show ( ) kinetics.
There are three recognized patterns of feedback inhibition in biosynthetic pathways. What are those?
Simple, Concertive, Cumulative
When the binding of one substrate molecule increases the affinity of the enzyme for a second substrate molecule or increases the rate of formation of product from sites already occupied. This is called ( ) cooperativity.
Positive cooperativity makes enzyme catalysis very ( ) to small changes in substrate level concentrations.
Covalent modification of enzymes or proteins occurs by the reversible attachment of chemical groups such as ( ) groups, ( ) groups, ( ) groups, ( ) groups, and ( ) groups.
Acetyl, Phosphate, Methyl, Adenyl, Uridyl
Sigma factors determine where RNA polymerase should bind.
The molecular weight of sigma32 is 32 kD.
Oxygen sensing proteins are all heme-containing proteins.
It is not the temperature per se that increases the activity and amount of sigma32, but rather the amount of ( ) protein.
The toxic forms of oxygen include ( two words ), ( two words ), and ( two words ), which can damage molecules such as DNA, proteins, and lipids.
Reactive oxygen species can oxidatively damage guanine to form ( ), which triggers GO system in bacteria.
The most common (& bad) outcome of DNA damage by UV light is (two words)
When E. coli cells are exposed to H2O2, OxyR is required to transcribe a set of genes for cellular defense and therefore will be activated. The active form of OxyR has an intramolecular ( two words ) between two cysteine residues.
List four functions of the E. coli heat-shock proteins:
1. Folding newly synthesized proteins at all temperatures
2. Export of proteins at all temperatures
3. Refolding of misfolded polypeptides
4. Proteolysis of improperly folded
Acidic environment is easier for ATP generation via ATP synthase, so it is optimal for bacterial growth.
Bacteria maintain their internal pH and osmolarity by controlling water permeation (blocking/allowing water permeation) through the cell membrane.
Turgor pressure has nothing to do with bacterial cell growth and division.
In bacteria, turgor pressure is generally exerted across the outer membrane.
No bacterium can live in 5 M NaCl environment.
In the presence of high salt concentrations inside bacterial cells, ionic interactions (e.g. between amino acids in a protein) are weakened.
Bacteria can be found growing in habitats that vary in pH from 1 to 11. However, regardless of the external pH, the internal pH is usually maintained within 1 to 2 units of neutrality, which is necessary to maintain viability. Thus, bacteria maintain a pH gradient (DpH) across the ( two words ).
The regulation of intracellular pH is mostly a consequence of controlling the flow of protons across the cell membrane. On the other hand, for bulk proton to take place, the pumping must be done ( ). For example, when the cytoplasm becomes too basic, protons are brought in via exchange with outgoing ( ) or ( ).
Electroneutrally, K+, Na+
In alkaliphiles, the use of Na+/solute symporters is advantageous because solute transport is driven by the ( two words ) rather than the ( two words ), the latter being low because of the inverted ( ).
Sodium Potential, Proton Potential,Δ pH
In acidophiles, the maintenance of the large DpH requires an ( two words ) at low pHout. In turn, this is due to an inward flux of ( ) greater than an outward flux of ( ).
Inverted Membrane Potential, K+, Protons
It looks like an acidophile has an advantage of having pre-existing proton potential, thus generating ATP through ATP synthase. But there is a problem without any compensatory mechanism. What is the problem?
It costs energy to maintain the gradient
An alkaliphile should maintain the pH condition above. Then, what is the main challenge for these bacteria?
Very little H+ outside the cell so it's difficult to make ATP
Mechanosensitive channels exist in the cell membrane. (T/F)
Mechanosensitive channels are proteins. (T/F)
Mechanosensitive channels proteins should have strong interaction with lipid molecules. (T/F)
SoxR and OxyR are involved in transcriptional regulation by sensing superoxide and hydrogen peroxide, respectively. Then, their activity is DNA binding. (T/F)
What could be the main job(s) of heat shock proteins in bacteria (well, let's say E. coli) when temperature is higher than normal condition?
Renature proteins or dispose of the denatured protein
Role of rpoD
Housekeeping sigma factor
Role of rpoH
Genes induced by heat shock
Role of rpoS
Genes for stationary phase
Role of rpoE
Genes for very high temperature
Role of rpoN
Genes for nitrogen metabolism
Role of mutS
Identify basepair mismatch problem
Role of mutL
Role of mutH
Add nick to DNA
A. - ("simple")
B. -/- ("concerted")
C. -/- ("cumulative")
If Km is high, this means: The enzyme has ( ) affinity to the substrate
Which curve (A or B) shows more sensitivity in terms of enzyme activity in response to change in substrate level?
A (sigmoidal curve)
1. Binding of + effector will (increase or decrease) Km of the enzyme.
2. Binding of - effector will (increase or decrease) Km of the enzyme.
1. decrease Km
2. increase Km
All the molecules below can be environmental signaling molecules in bacteria. Which one will penetrate cell membrane freely?
c) Inorganic phosphate
d) N-hexanoyl homoserine lactone