Quiz 2 Microbiology Lab BIOL 3101 Spring 2011 125 points

1. Match the following phrases with the terms below that apply to them. Phrases can be
used once, more than once, or not at all. Write the appropriate letter (s) after the term.
Phrases: a) Killed non-transformants;
b) Used to make chromosome fall out of solution
c)Brings the pH down;
d) Protects DNA from self degrading;
e)Dissolves membranes during cell lysis;
f) Punches holes in cells during a transformation;
g) Plant starch;
h) pHUG21 contains this;
i) Degraded by a -lactamase;
j) Forms ionic bonds with DNA
k) Nucleases require this;
l) Forms H bonds with water;
m) Found in TE;
n) Binds nonspecifically to DNA and RNA (36 points)
Terms:
1) CaCl2 f
6) detergent e
2) ampicillin a, i
7) EDTA m
3) neutralization buffer b, c
8) OriC h
4) ethanol l
9) Tris d, m
5) glycogen n
2. Answer the following questions (26 points):
a. What is the major question we are asking in the molecular biology labs? (6)
Are hugWXZ required for heme transport?
b. Write the name of the gene that encodes the outer membrane heme receptor from
Pleiomonas shigelloides. It must be written correctly to receive any credit. (5)
hugA
c. How is P. shigelloides like enterotoxigenic Escherichia coli? (5)
It causes a cholera like disease.
d. Describe the chemistry behind neutralization buffer and how it achieves its effect.
Make it clear what procedure it is involved in and how it makes that procedure work. (10)
It brings the pH down, which disrupts interactions between polar molecules and water.
The chromosome, which is large then precipitates and the plasmid which is small remains
in solution during a plasmid isolation.

3. Paragraph correction questions. 15 points

_________________________ Our plasmid isolation was done so we could isolate the
plasmid from the strain in which the plasmid was constructed and then move the plasmid
into DHE-1, a heme biosynthesis mutant. DHE-1 also is a siderophore synthesis mutant,
as a kanamycin resistance gene interrupts one of the genes encoding a protein involved in
making a siderophore. A siderophore is secreted into the environment and binds
elemental iron and then the iron and the siderophore are transported into the cell. DHE-1
is not ampicillin resistant until one of the plasmids we are working with is moved into the
strain. When pHUG17.1 is added to competent DHE-1, the cells are then kept on ice
overnight and then heat shocked for a short period (to close the holes in the cells). Then
they are incubated for an hour in L broth with ampicillin (ALAl) to allow the cells to
recover and grow.
_________________________ In order to cause disease, an intestinal pathogen must
survive the acidity of the stomach. Vibrio cholerae is much better (worse) at this than
Shigella dysenteriae. Enteroinvasive Escherichia coli causes a disease comparable to
the disease caused by S. dysenteriae. Both organism get into cells, multiply, lyse the cell
and spread to other cells. If an organism does not deal with stomach acidity, the number
of bacteria that you would need to consume to get sick would be high. The ability to
acquire iron is also important for a pathogen, and the iron in the body is normally bound
to various proteins. Most of our cells contain heme because it is present in hemoglobin
(cytochromes).
4. Fill in the blank with the best answer. Spell out names fully if they were given to you
in lab. 48 points
competent____________ Capable of taking up DNA.
EDTA______________ Chelates magnesium.
EDTA_______________Chemical that protects DNA from nucleases during plasmid
isolation.
70% ethanol________ _Used to wash the nucleic acid pellet at the end of an
ethanol precipitation.
_RNA______________This type of nucleic acid is single stranded and would be found
in the pellet of our plasmid isolation after the ethanol
precipitation.
TonB-dependent_______The two types of transport systems involved in Plesiomonas
ABC transport____________shigelloides heme transport.
Heme ______________These two substances move through inner membrane proteins
protons_________________involved in P. shigelloides heme transport.
_Staphylococcus aureus_Methicillin was covered previously when we discussed this
organism which is sometimes resistant to it.
_penicllin G__________This -lactam antibiotic is not appropriate for use in Grambacteria because it cannot get through the outer membrane.
Sodium acetate________We need to get rid of this substance from the pellet as one of the
final steps in an ethanol precipitation.