Examen Parcial – PrácticaPreguntas Orales – Hay 8. They will c.docx

Examen Parcial – Práctica
Preguntas Orales – Hay 8. They will come from the “Practice
preguntas orale”s list
Actividad auditiva A
Verbos reflexivos.
* pronombre reflexivo + el verbo
Conjuga levantarse:
yo
nosotros
tú
vosotros
usted/él/ella
ustedes/ellos/ellas
Conjugate the reflexive verbs according to the subjects. OJO:
Don’t forget to use the correct reflexive pronoun.
Todos los días Lucia _____ __________ (acostarse) a las 10 de
la noche.
Yo _____ __________ (quitarse) la ropa antes de ducharme.
¿Tú _____ __________ (ponerse) la ropa antes de cepillarte
los dientes?
Los hijos _____ __________ (despertarse) a las 9 de la
mañana.

Los verbos como gustar.
Los verbos como “gustar” : dos conjugaciones:
Usamos los pronombres de objeto indirecto (me, te, le, nos, les)
para indicar a quién se refiere el gusto
· gusta = se refiere a una cosa singular o una actividad (verbo
infinitivo)
· Me gusta la manzana.
· ¿A ti, te interesa esquiar?
· gustan= se refiere a una cosa plural
· Me gustan las manzanas.
· A los estudiantes, les gustan los deportes.
Llena los espacios con la conjugación correcta del verbo como
gustar.
1. ¿A ti ________ _____________ (gustar) las vacaciones de
primavera?
2. A mí, ________ _____________ (interesar) los museos
arqueológicos de México.
3. A Pedro y Marta ________ _____________ (encantar)
viajar por América Central
4. A mi tía Paula ________ _____________ (interesar) las
costumbres de la gente indígena.
5. A nosotros ________ _____________ (importar) sacar
buenas notas.
7. Pero a mí, ________ _____________ (encantar) todas las
playas de México.

8. A Uds. ________ _____________ (gustar) tomar el sol en
Acapulco?
Palabras afirmativas y negativas
algo ----- nada
alguien ----- nadie
algún ----- ningún
alguno ----- ninguno
siempre ----- nunca
también ----- tampoco
o…o ----- ni… ni
Change the afirmative sentences to negative and the negative
sentences to afirmative. *Don’t forgeto to use the doublé
negation when necessary.
1. A todos les gusta comer pasas.
2. Elene siempre pide alguna bebida.
3. Ningún estudiante va a comer pizza.
4. Alberto va a pedir paella también.
5. Yo quiero comer algo antes de salir de casa.
6. Juan no quiere comer carne de res tampoco.
7. Jamás viviría ni en Alaska ni en Canadá.
Responde a las preguntas con por lo menos una palabra negativo
o afirmativa.
¿Conoces personas que son de
Rusia?_______________________________________________
______
¿Vas a visitar México y Panamá el próximo
semestre?_______________________________________
¿Compras libros cuando vas a la
librería?______________________________________________

____
Comparaciones:
Desigualdad:
más/menos + adjective + que
más/menos + noun + que
más/menos + adverb + que
verb + más/menos que
Irregulares:
mayor / menor + que mejor / peor + que
Igualdad:
tan + adjective + como
tanto/a/o/as + noun + como
verb + tanto como
Write comparisons in complete sentences.
1.Alfredo / tener / Coca-Cola / Graciela
2.Graciela / tener / cuadernos / Alfredo
3.Alfredo / tener / bolígrafos / Graciela
4.**Graciela / ser / (edad [age]) / Alfred

Haz una comparación de igualdad y una comparación de
desigualdad entre la biblioteca, el cine y el hotel. (palabras
útiles: alto, bajo, grande, pequeño, ventanas, puertas)
desigualdad:
_____________________________________________________
______________
igualdad:
_____________________________________________________
________________
Superlativos
EL/LA/LOS/LAS + sustantivo + MÁS/MENOS + adjetivo +
DE + sustantivo/ verbo
Superlativos. Add the necessary words to write a superlativo.
Maimi / ciudad / Estados Unidos
_____________________________________________________
____________________
Juanes y Shakira / cantantes / mundo
_____________________________________________________
____________________
El fútbol / deporte / mundo hispano
_____________________________________________________
____________________
Yo / estudiante/ CU Denver
_____________________________________________________

____________________
Amanda/ persona / nuestra familia
_____________________________________________________
_________________
Los pronombres de objetos directos
me
te
lo
la
nos
los
las
Respond to the question and replace the direct object with the
pronoun in the answer.*The questions and responses are in the
preterit
Modelo: ¿Compraste un refrigerador?
Sí, lo compré. OR No, no lo compré
¿Hiciste la tarea anoche?
___________________________________________________
¿Compraste un
abrigo?______________________________________________
_______
¿Usaste el secador de pelo esta mañana?
____________________________________________

¿Comiste las papas fritas hoy ?
___________________________________________________
Los pronombres de objeto indirecto
me
te
le
nos
les
Fill in the spaces with the correct indirect object.
1. Luis, (a mi) _______ explicas la gramática por favor.
2. ¿Por qué no (a ella) ________ preguntas a la profesora?
3. ¿Quieres ayudar________ (a nosotros) a estudiar la
gramática?
4. _______ (a él) voy a preguntar después de clase.
5. Sí, ¿cómo puedo ayudar_______ (a ellas)?
6. ¿Por qué no (a nosotros) _______ preguntas algo?
7. Yo (a ustedes) _______ pregunto algo en español y ustedes
(a mi) _______ contestan en inglés.
Pronombres dobles.
First, circle the direct object. Then underline the indirect object.
Finally, re-write the senences with the correct pronouns.
OJO* OI first, OD second

Le hiciste la cama a tu hija.
_____ ______hiciste.
Gregg te compró unas tostadoras este fin de semana
_____ _____compró.
Mi madre me lavó la ropa
____ ____ lavó.
La profesora les hizo preguntas a los estudiantes.
_____ _____ hizo.
El pretérito – regulares e irregulares.
Conjuga los verbos regulares en el pretéritoHABLAR COMER
yo
tú
usted/él/ella
nosotros

vosotros
ustedes/ellos/ellas
Los verbos irregulares:
Diferentes en sólo en el YO Verbos que terminan en..
-car - qué
-gar - gué
-zar - cé
I - Y en 3ª persona singular y plural
Verbs with 2 vowels together
Verbos que terminan en –UIR
E-I & O- U en 3ª persona singular y plural
Verbos de –ir con cambio de raíz en el presente
Verbos irregulares con raíz de U
J- stem
I-stem
*all don’t have accents
Totalmente irregulares:

El sábado a las diez de la mañana, Neil ___________(limpiar)
su casa.
Anteayer tú ___________(estar) en una discoteca hasta las tres
de la mañana.
Mis amigos y yo __________________________ (patinar) dos o
tres veces allí.
El lunes pasado yo _______________(dar) una caminata con mi
amiga.
Yo _________________ (correr) cerca del teatro.
Marilyn no____________(venir) al museo ayer.
A la una yo _________________________(llegar) al partido con
mis amigos.
¿ __________________________ (abrir, tú) el regalo que te di?
Ayer Uds. __________________(visitar) un museo en Denver.
Anoche todos nosotros _________________ (ponerse) la
chaqueta porque estaba lloviendo.
¿__________________ (tú, traer) el libro de español a la clase?
Después, nosotros __________________ (comer) cerca de un
bar.
Esta mañana no __________________ (cepillarse) los dientes!
El fin de semana pasado Colin _____________(hacer) un picnic.
A las cuatro, Bryce _________________ (barrer) el piso de su
apartamento.
El sábado por la tarde, yo les_____________ (pasar) la
aspiradora a mis compañeras de cuarto.
¿_______________(tú-traer) la cafetera?
El mes pasado los jugadores __________________(ganar) dos
partidos de béisbol.
¿ __________________________ (hablar, tú) con tu madre
hoy?
El año pasado Christina __________________(leer) un libro
sobre el equipo de Colombia.
¿Tú __________________ (conducir) a la universidad esta
mañana?

Mis padres __________________________ (vender) sus
coches.
Javier __________________________ (quitarse) la chaqueta
cuando salió el sol.
Él le ___________________ (decir) la verdad al juez.
El viernes pasado Alex y Shawn ____________(ir) a un
concierto de Shakira.
El Profe ________________ (tener) que leer todas las
composiciones el sábado.
El cliente le __________________ (pedir) una cerveza al
mesero.
Yo__________________ (pagar) cien dólares por el suéter.
Anoche yo _______________(poner) la mesa antes de cenar.
LECTURA –viene de Capitulo 9
COMPOSICIÓN
Describe tu última Navidad o vacaciones. ¿Dónde fuiste?
¿Cómo viajaste? (medio de transporte) ¿Qué hiciste mientras
estuviste allí? ¿Cuál fue la cosa más divertida que hiciste y por
qué? ¿Cuándo regresaste? Escribe por lo menos 8 oraciones. (10
puntos)
Rubrica:
Content & Grammar - 8
Cohesion, Transitions, Logic - 2

1
MMG 408. EXPERIMENTS I - III
Experiment I: Bacterial Growth and Induction of Bacterial
Operons, Bacterial Genetics
Exponential Growth and Induction of the E. coli lac Operon
All types of bacteria multiply by binary fission so their growth
is exponential. As a
consequence, if the number of bacteria in a growing bacterial
culture is plotted versus the time of
growth on semilog graph paper, a straight line will result. The
optical density (OD) of the culture is
due to light scattering by the bacteria in the culture and is
proportional to the number of bacteria in the
culture.
To conserve energy, cells often only express genes that are
required for maximum growth under
the conditions the cells are in. For example, they will only
express genes whose products are required
to use a particular carbon and energy source if that carbon and
energy source is present in the medium.
Then, and only then, will they induce the transcription of the
genes to use that carbon source.
The genes of the lac operon are the classical example of
inducible genes. This operon consists
of a gene whose product transports lactose into the cell (lacY)
and a gene whose product degrades
lactose to glucose and galactose (lacZ). The product of the lacZ
gene is the enzyme, β-galactosidase,

that can be easily assayed using the β-galactoside, ortho-
nitrophnyl-galactoside (ONPG), or 5-bromo-4-
chloro-3-indoyl-β-D-galactoside (X-Gal) which turn color when
cleaved by β-galactosidase.
Note: This information is provided to you so that you may have
a rudimentary overview of the
experiment you are about to perform. You may reference this
information in your reports but
2
must cite this work (even if you reference it in your methods).
You may not simply rephrase
these background sentences as that would constitute plagiarism
just as simple copying would.
3
Experiment I: Day 1 (9/1) Using Escherichia coli Mutants to
Understand the lac Operon
1. Take two minimal, Histidine, Tryptophan, Arginine, X-Gal
plates and divide them into thirds with a marker.

2. Using your spread plating technique, spread 100µL of the
inducer (100mM IPTG) on one plate.
3. Streak (for isolation) each of the three mutants; E. coli
CB2881 (lac+), E. coli PK191 (lacZ-), and E. coli
CB846 (lacI-) in one third of each plate.
Complete the accompanying worksheet for Experiment I: Day 1.
It should be included in your report.
Follow-Up
1. Record your observations.
Did each mutant behave as expected? Why or why not? Can you
continue using the E. coli CB2881?
4
Experiment I: Day 1 Worksheet. Name:
To be completed during the lab period. This and all worksheets
must be stapled with your report.
1. What is x-gal in this experiment? What is the mechanism of
color change?

2. Briefly explain the action of IPTG.
3. One of the Escherichia coli mutants is always producing β-
galactosidase.
What is the term for this type of expression?
4. Record your color predictions below.
-IPTG +IPTG
E. coli CB2881
(lac
+
)
E. coli CB846
(lacI
-
)
E. coli PK191

(lacZ
-
)
E. coli CB2881
(lac
+
)
E. coli CB846
(lacI
-
)
E. coli PK191
(lacZ
-
)
First result that needs an explination
change into blue
blue
blue
5

Experiment I: Day 2 (9/6): Generating a growth curve for
Escherichia coli CB2881
Working in pairs.
An overnight culture of E. coli CB2881 has been diluted for you
to an OD625nm of approximately 0.05 (likely
1:50).
1. Remove one 5ml tube from the 37°C shaker and label it with
your name.
2. Blank a spectrophotometer with sterile LB at 625nm (Note:
mark the top of your tubes and blank using
the same side each time).
3. Measure and record the optical density (O.D. 625nm) of your
freshly diluted culture (Note: mark the top
of your tubes and read the same side each time).
4. Return the tube to the shaking incubator.
5. At 30 minutes intervals, record the O.D. 625nm of the
culture.
6. At 90 minutes, and 120 minutes, remove a 20µl sample for
viable cell counts.
7 To do this, place 180 µl sterile saline solutions into each of
the 7 microfuge tubes.
8 Add 20 µl of the bacterial culture to the first, mix and transfer
20 µl to the next tube.
9 Continue this operation until the last tube. Remember to
change tips at every dilution!
10 Using spread plate technique; plate the last 3 dilutions on LB
agar (using 100 µl on each plate).
Preparation for Experiment II Selection of rifampin resistant

mutants (rpoB)
Note: This activity will be reported in the lab report for
Experiment II. You will need to record your methods
and data in your notebook as usual, but will not be reporting on
them until Experiment II is completed.
1. Centrifuge 1ml of the overnight Escherichia coli HR171
(rifS) in a microfuge for 1 minute.
2. Decant the supernatant and resuspend the cells in the LB that
remained on the walls of the tube after
decanting (around 100-200 µl only).
3. Spread the whole volume on an LB with rifampin plate.
4. Incubate at 30°C.
fida
fida
results:I got 40 colonies in this plate which was success…we
restreaked it again for further study
dillution10^-610^-710^-8
At t90197 211
t120 226 242
OD at 0t was 0.185

at 30min was 0.206
at 60 min was 0.358
at 90min 0.528
at 120 min was 0.656
6
Experiment I: Day 3 (9/8)
Follow-up of determination of growth curve:
1. After 1-2 days of incubation, count the number of colonies on
your plates.
2. Back-calculate the concentration of viable cells in the culture
at each of the sampling points.
3. Plot the data as O.D. 625nm vs. time (minutes) and as colony
forming units/ml versus time on the semi-log
graph paper.
Preparation of buffer for β-galactosidase assay
Each student calculates how to prepare 100 ml solution of a
buffer of the following composition:
Na2HPO4 H2O 60 mM
NaH2PO4 H2O 40 mM
KCl 10 mM
MgSO4 1 mM
di-thio-Threitol 30 mM
CETAB 0.15%
pH 7.0.

Check the calculation before preparing the buffer among your
colleagues and let the instructor approve it.
Prepare the buffer in groups one per bench.
Note: Since weighing small quantities has a considerable error
we will provide you with 0.2 M solutions of the
ingredients.
Measuring the Induction of the Escherichia coli lac Operon
Your instructor has already diluted an overnight culture of E.
coli CB2881.
1. Take 5-6 ml from the shaking incubator (250 rpm at 30°C)
and label it with your name.
2. Check the OD625 nm using the spectrophotometer. Blank
with LB.
3. Continue incubating the culture until it reaches an OD625 nm
of 0.3 (takes about 2 hrs to reach O.D. of
0.3).
4. During this period prepare the assay by adding 0.5 ml (i.e.
500µl) of assay buffer (prepared fresh) to
four 13x100mm tubes.
5. Number the tubes 1-4. Keep tubes on ice.
6. When the culture reaches the target OD (0.3), add 0.5 ml to
Tube 1. Mix well, leave on ice.
7. Add 50-52µl of IPTG to the remaining cells and continue
shaking (Note: Don’t confuse IPTG (inducer)
with ONPG (substrate)..
8. After adding the inducer record the OD625 nm at 15, 30, and

60 minutes, and add 0.5 ml of each time
sample to Tubes 2 (i.e. 15 min.), 3 (30 min.) and 4 (60 min.),
respectively, kept on ice.
9. Begin the β-galactosidase assay.
10. Vortex each tube for at least 10 seconds to permeabilize the
cells.
11. Warm the tubes for a few minutes in the 30°C water bath.
12. Add 200µl of ONPG to each tube and return the tubes to
30°C water bath. (Note: It helps to stagger the
additions so that you have time to manipulate each tube. For
example: make additions of ONPG at 30
seconds intervals).
13. Start timing the reaction when you add ONPG and place the
tubes at 30°C.
7
14. When tube #4 turns yellow (usually takes 15’), record the
actual reaction time.
15. Begin stopping the reactions by adding 1.5 ml of 1M
Na2CO3 (at the same 30 second intervals). Note:
Do not keep Na2CO3 on ice or it will come out of solution.
16. Using the spectrophotometer record both the OD420 nm and
the OD550 nm. Blank with Assay buffer &
LB (1:1). The first of these measurements determines the
intensity of yellow color due to conversion o
ONPG to nitro-phenol, the second determines the turbidity of

the mixture due to the bacterial cell
suspension.
17. Calculate the specific activity for each tube.
Continued Preparation for Experiment II Selection of rifampin
resistant mutants (rpoB)
Note: This activity will be reported in the lab report for
Experiment II. You will need to record your methods in
your notebook as usual, but will not be reporting on them until
Experiment II is completed.
1. Count the number of colonies grown on your rif plate.
2. Streak out two rifR mutants on LB rifampin plates to purify
[these came from plating E. coli strain
HR171 rifS on rifampin plates (see experiment I, day2), looking
for spontaneous mutants).
3. Incubate at 30°C overnight.
results:1
at :0t OD was 0.17
after one hour the OD was 0.388 then we add IPTG To the tube
after 15 min was 0.495
after 30min was 0.604
after 60 min was .778
result2……………………………..
OD420OD550
Tube1.128nm.083
2 .29.091
3 .526.099
4 1.33.190

8
Questions to answer in your final report on Experiment I
1. What is the generation time of your bacterial culture?
2. How many viable bacteria per ml would be in your culture
when the OD625 nm reaches 1.0?
Plot growth both in OD625 nm vs. cells/ml and vs. time.
3. Why do the colonies of a wild type E. coli strain on the X-gal
plate turn blue only when IPTG is also on the
plates?
4. Why is the lacZ mutant never blue on X-gal? The lacI mutant
always blue even without IPTG?
5. Why did it take some time after IPTG was added for the β-
galactosidase in the cell to reach its maximum level
in units/mg protein?
6. What is the approximate specific activity (units per mg
protein) of β-galactosidase in your culture each time
before and after adding IPTG? Also express per cell. What

would you have observed if you had done the
induction with E. coli CB846? E. coli PK191?
€
Units
mgprotein
=1000 ×
OD420 −1.75 × OD550
t × v × OD625 × 0.2
$
%
&
'
(
)
t = reaction time in minutes (the length of time you incubated
the cells in ONPG)
v = volume of cells you added per ml of reaction mix
For example: if you added 0.2 ml cells to 2ml assay buffer, it
would be 0.1.
(Since you’ve added 0.5 ml cells to 0.5 ml assay buffer, then
it should be 1.0)

OD625 nm = the OD the cells would have had at the time you
removed them from the culture (OD ≈ 0.3?).
We are assuming that 1ml of cells at OD625 nm = 0.5 has
100µg protein.
7. If the OD625 nm = 0.2 at the time IPTG was added, what
would it be 1 hour later?
Refer to your growth curve.
9
10
make a graph here with 2 lines
one for the Optical density and one for generation time or log to
cell number/ml
so take the time at X axies(0-30-60-90-120..)vs OD at the Y
axies from the left and the log to cell number to the left and
describe it using this resultsdillution10^-610^-710^-8
At t 90 19721 1
t120 22624 2

growth rate calculation must be shown so you have to
take197*10^6 and calculate =cfu/ml
21*10^7
1*10^8=cfu/ml and so on
11
Experiment II: Gene Mapping by Homologous Recombination
In this experiment you will isolate a rifampin resistant mutant
of Escerichia coli HR171. The rifR
mutants occur spontaneously in a growing culture and will be
selected on a rifampin agar plate. Once the
desired mutant is isolated, you will map the mutation by
conjugation and transduction.
The E. coli chromosome is a circular DNA molecule about 4 x
106 base pairs in length. Normal E. coli
is haploid; it contains only one copy of each of its genes. In
order to map the order of genes on the E. coli
chromosome, one measures the frequency of recombination
among them using genes with differing alleles (i.e.
a mutant allele like rifR and a wild type allele like rif
S
). Since the organism is haploid, for recombination to
occur one needs to transfer at least part of the chromosome of
one strain (called the donor) into a cell of the
other strain (the recipient). Experiment II uses two different

methods to do this - conjugation and transduction.
During conjugation DNA is passed from one bacterial cell to
another by a conjugative plasmid.
Normally, the plasmid only transfers itself but if it has
integrated into the chromosome (an Hfr strain) it will
also transfer the entire chromosome because the chromosome
has become an integral part of the plasmid. We
can use Hfr strains for mapping mutations because the farther a
gene is on the DNA from the site of integration
of the plasmid, the less frequently the gene will be transferred.
Transduction is the transfer of DNA by a bacteriophage;
bacterial DNA is packaged in a phage head and
then enters another bacterium upon subsequent infection of this
bacterium. We can use transduction for
mapping because the phage head will only hold a small piece of
bacterial DNA so for two genes to both be
carried in the same head they must be close to each other in the
DNA. The closer together they are, the more
often they will be packaged together and cotransduced. Also,
the frequency of the various recombinant types
among the transductants will depend upon the order of the
genetic markers.
12
Genetic map of the Escherichia coli chromosome. The numbers
refer to map position in minutes (minutes
required for the gene to be transferred in conjugation), relative

to the thr locus. 52 loci are shown, chosen on
the basis of utility in mapping studies. Inside the circle, the
leading transfer regions of a number of Hfr strains
are indicated. The large arrow points at KL16, the transfer
region of the Hfr strain employed in this experiment.
The map positions of mutations of E. coli strains HR171 F-
hisG argH trpA strR (rpsL), KL227 metA, and
KL16-99 recA thi strS are shown with arrows. The region
deleted in strain PK191 Δ(proB-lac)XIII is indicated
by the dashed line.
strR
13
Experiment II Transduction: Day 1 9/13 Preparing P1Cm c1-100
Lysogen
In the following order:
1. Place 100µl of an overnight culture of E. coli KL227 metA
into a 13 x 15mm test tube.
2. Add 5 µl of 0.1 M CaCl2.
3. Add 50 µl of phage P1 c1-100 suspension provided.
4. Incubate at 32°C for 10 min to allow for phage adsorption

and expression of CmR.
5. Plate 20 µl on LB – chloramphenicol (Cm 25 µg/ml) and
incubate at 30°C.
Note: Incubation at higher temperature will induce the phage to
lyse the cells!
Experiment II Conjugation: Start Cultures of Donor and
Recipient
1. Inoculate 2ml of LB with one colony of Hfr KL16 (donor).
2. Inoculate 2ml of LB-rif with one colony of your mutant rpoB
(recipient).
Note: Try to choose a larger, faster growing colony.
2. Incubate in the 37°C shaker at 200rpm.
COLONIES were small and around 16 colonies
14
Experiment II: Day 1 Worksheet.
To be completed during the lab period.
1. What is a lysogen?

2. Why do the agar plates include chloramphenicol?
4. Why do we incubate the plates at 30°C and not 37°C?
Name:
Lysogen: A bacterium with phage integrated into its genome. In
such a state the replication of the phage DNA is under the
control of the host and the phage's own replication system is
repressed (stable lysogen). Rarely does the suppression of the
replication of the phage fail after insertion (abortive lysogeny)
and the prophage not replicated and lost by dilution during
subsequent divisions of the host.
The incubation of bacterial culture plates are done at 30 degree
but not at 37 degree because in that way we do not allow the
pathogens which might be harmful for the human to grow in the
plates.

Chloramphenicol is a broad spectrum antibiotic usually used in
eye drops. In bacterial culture, this antibiotic is often used to
amplify the final plasmid concentration if we use a bacteria
having a low copy number plasmid. Chloramphenicol helps in
improving plasmid quality in following ways:
The host bacterial culture is exposed to the antibiotic
chloramphenicol, which inhibits bacterial protein synthesis.
This leads to inhibition of chromosomal replication (because
this also relies on ongoing protein synthesis) and inhibition of
cell division.
Plasmids, only requiring proteins that are more long-lived,
continue to replicate even though chromosomal replication and
cell division has stopped.
Eventually plasmid replication will stop when the cell becomes
exhausted (proteins used up) but the average copy number will
have increased significantly.
15
Experiment II Conjugation: Day 2 9/15) Hfr Cross
1. Dilute each culture (donor and recipient) 1:5 in fresh LB
[(i.e. 1ml culture + 4ml LB, (2 tubes)].
2. Label three 13x100mm tubes.
3. In tube 1 mix 500µL E. coli Hfr KL16 (donor) and 1ml of
rpoB mutant (recipient).
4. In tube 2. dispense 500µL of E. coli Hfr KL16
5. In tube 3 dispense 1ml of rpoB mutant.
Tubes 2 and 3 are your controls of the donor and recipient,

respectively.
6. Incubate without shaking for 90’ at 37°C in a water bath.
7. During incubation prepare M9 streptomycin plates by
spreading 50µL of each amino acid as shown
below.
Note: You must take care not to cross-contaminate the
spreader. Rinse with the tap water squeeze bottle
between amino acids.
6. After incubation add the cultures to three separate microfuge
tubes.
7. Centrifuge (max. speed) for no more than 30 seconds to
pellet the cells.
8. Decant the supernatant carefully!
9. Resuspend the cells in 350µl of saline (repeat steps 7-9, in
order to wash the cells from residual LB).
10. Spread the 100µl on each of the following plates:
11. Incubate plates at 37°C.
Experiment II Transduction: Day 2 Continue propagation of
cultures.

1. Streak out your rpoB mutant on a LB-Rif plate (from liquid
culture).
2. Streak your P1Cm c1-100 lysogen on a fresh LB Cm plate
and incubate at 30°C (from plate).
53 something went wrong and it should have no growth
non
170
311colonies
62 colonies
16
1. Why do we incubate conjugation mix without shaking?
3. When you decant something, what are you doing?

Name:
Decanting in laboratory procedure means to separate out the
sediment from the supernatant. When we mix one preparation by
centrifugation, the mixture separates in two distinct layers- an
upper clear liquid layer which is known as supernatant and the
bottom solid layer which is the mixture we need for further
processing. Decanting means to slowly and gradually separating
the upper supernatant from the bottom solid precipitate so that
we have only the precipitate which is required for further
processing.
The conjugation of bacteria requires the formation of sex pilli
which helps the baceria to hold another to transfer the genetic
material from one another. If we shake the conjugation mix, that
will not help the sex pillis to form or if already formed, the
shaking will destroy them. That is why it is not recommended to
shake the conjugation mix while incubating them.
17
Experiment II Conjugation: Day 3 9/20 First Analysis of
Transconjugants
Work in groups of 4 so that each group has at least 40
transconjugants to analyze.
1. Examine the controls.

2. Assess the purity of all the plates.
3. Count the number of transconjugants on each of the selective
plates.
4. Record your counts on the class spreadsheet.
5. Prepare 8 M9 streptomycin plates with trp and arg as before
per group.
6. Purify 48 of the His+ conjugational recombinants.
Note: Pick isolated individual colonies only.
7. Save the plates with the P1 lysogen and of your rpoB mutant
in and store them in the refrigerator for the
next day.
since the mixture plate has much growth 311 colonies we are
gonna streak them again
18

Experiment II Conjugation: Day 4 9/22 First Analysis of
Transconjugants
1. Spread the appropriate amino acids to prepare the following
plates:
2. Draw a line on the upper center of your petri dishes with a
Sharpie.
3. Use the paper templates provided to place a small (3 – 4 mm)
streak
each of your colonies on each plate.
6. Do this using toothpicks or wires. Touch an isolated colony
and patch onto each plate in this order:
Experiment II Transduction: Day 4 Prepare Overnight Culture
of P1 Lysogen
1. Inoculate a single, isolated colony of the lysogen into 5 ml
LB.

2. Incubate at 30°C.
we have been patcheed the 16 colonies and also the new
streaked plate because we did not get enough in the patching
plate to get 50 patches total
50
2 grew
50
5
19
1. What is the purpose of patching each of these plates?
Tryptophan Only:
Arginine Only:

Rifampicin Only:
Tryptophan and Arginine:
2. Why are we careful to patch onto the tryptophan and arginine
plate last?
Name:
20
Experiment II Transduction: Day 5 (9/27) Induction of the
P1Cm c1-100
An overnight culture of the P1 lysogen of E. coli KL227 metA
has been diluted for you.

1. Remove one tube from the shaker and label it as your own.
2. Check the OD625 nm.
3. Continue to incubate with shaking at 32°C.
4. Check the OD625 nm periodically until it reaches at least
0.6-0.7.
5. Transfer P1 lysogen culture to 42°C water bath shaker and
shake vigorously for 30 min.
6. Transfer to 38°C and shake vigorously for 1.5 to 2 hours or
until a lysis is detected.
Note: If there is time, you may proceed through steps 1-4 for
Day 6.
7. Store phage stock at 4°C.
Experiment II Conjugation: Day 5 Analysis of Transconjugants
1. Observe your patched plates.
2. Record the result from your Hfr cross and enter into a
spreadsheet with class results.
Follow-Up
3. Construct a “gradient of transfer graph” from your results and
the pooled class results.
Note: Use pages 206-207 of Snyder and Champness 2nd
edition (pp. 179-180 of third edition) as a guide.
OD starts at .434
.663 at 32c
.983 at 42c

after30 min was .725
15min was .713
15min was .662
15min was .531
15min was .381
and then we saw alot of small dots forming which means it’s
lysed
21
Experiment II Transduction: Day 6 (9/29) Titration of Phage
8. Vortex your culture and check the OD625 nm to confirm the
cells have lysed.
9. Add a drop of chloroform to your P1Cm c1-100 and vortex.
10. Transfer 1.5ml to a microcentrifuge tube.
11. Centrifuge for 2 minutes.
12. Transfer the supernatant to a fresh microcentrifuge tube.
13. Add another drop of chloroform and vortex.
14. Spin again for 1 minute.
15. Use 20 µl of the supernatant to serially dilute to 10-4, 10-5
and 10-6.
To do this:
16. Place 180 µl sterile saline solution into each of the 6
microfuge tubes.
17. Add 20 µl of the phage suspension to the first, mix and
transfer 20 µl to the next tube.
18. Continue this operation until the last tube. Remember to
change tips at every dilution!

19. Save the remaining lysate at 4°C.
20. Titer the phage to determine the number of phage particles
in your lysate:
To do this:
1. Place 100 µl of fresh culture of E. coli KL227 in 3 x 15 ml
tubes. Add 10 µl CaCl2 (from a100mM stock
solution, before adding 100 µl of your phage).
2. Add 100 µl of P1Cm c1-100 dilutions 10-4, 10-5, 10-6 to
individual tubes.
3. Incubate the tubes at 30°C for 10 minutes for adsorption of
the phage.
4. Label 3 R-Base agar plates.
5. Only when you’re ready. Add 4ml of melted R-top agar to the
tubes with bacteria – phage dilution tubes.
6. Mix the tubes by rolling them between your palms.
7. Pour the melted agar onto the plates. Act fast before the agar
has a chance to solidify!
8. When the agar has solidified, invert the plates as usual and
incubate at 40°C.
Prepare your rif (rpoB) mutant for transduction:
Inoculate 2ml LB rif with rifR (rpoB) mutant and incubate at
37°C with shaking overnight.
OD was .143
on the plate with 10^-6 dillution we got 400 pfu
on 10^-7 we got 77pfu
on the 10^-8 we got 12 pfu
so we picked the 77 to do the calcultion ,show the calculation

for the next step down
22
1. Why did we grow the lysogen and lyse the cells last lab?
(What were we trying to obtain?)
2. Why do we add chloroform to the lysate?
4. Why are we adding E. coli KL227 to the lysate tubes for
titration of the phage?

5. Why are we plating three dilutions of phage for titration?
6. Why do we need to calculate the titer?
Name:
23
Experiment II Day 7 (10/4): Transduction
1. Count the plaques on your titration plates.
Note: P1 plaques are very small and not easy to distinguish; use
magnifiers if necessary.
2. Calculate the titer of the phage using the dilution you trust
most.

3. Put 200 µl of the rifR (rpoB) mutant of E. coli HR171 his-
argH- trp- into 2 microfuge tubes.
4. Add 108 PFU to one tube.
5. Add 100 mM CaCl2 to a final concentration (f.c.) of 5 mM
Note: You should use the C1V1 = C2V2 equation to determine
the volume of calcium chloride to add. You
will need to add these volumes based upon the unique volume of
phage you already added. The tubes will
have a different V2; right?!
6. Incubate tubes at 30
oC for 10 minutes.
7. Prepare two plates:
8. Spread 100 µl each trp, his, met, on both minimal media
plates.
9. Add sodium citrate to tubes a f.c. of 10mM.
Note: You should be able to calculate this now. Be sure you
check C1.
10. Centrifuge tubes for 20 seconds.
11. Decant the supernatant.
12. Resuspend in remaining supernatant.
13. Spread the whole volume on plates.
14. Incubate the plates at 30°C.
in the control plate we got no growth
in the plate with the phage we got 5 colonies and the whole
class got a few colonies =explain
24

1. The tube without phage is a control for what?
2. Why do we not use arginine on the plates?
3. Next lab we’ll patch the transductants on minimal media with
different additions.
Which plate is the permissive plate?
4. Given that sequencing of the E. coli genome is cheap and

easy, what is the value in learning of these gene
transfer techniques? Or, aside from mapping, what are other
uses of conjugation and transduction?
Name:
25
Experiment II Transduction: Day 8 (10/6) Analysis of the
Transductants
1. Using a toothpick patch the transductants as follows:
2. Incubate the plates at 30°C.
each one per group patched it’s own colonies in one plate per
group since each one got a few colonies

26
Experiment II Transduction: Day 9 (10/11) Analysis of the
Transductants
1. Record and analyze your transduction data as well as the
pooled class data.
2. Perform the 3-point cross analysis.
Note: KL 227 (donor) = argH+ metA- rifS
rifR mutant of HR171 (recipient) = argH- metA+ rifR
Recombinant Phenotype Number of Recombinants
arg+ met+ rifR
arg+ met+ rifS
arg+ met- rifR
arg+ met- rifS
Draw the recombination crossovers to help you determine the
order of arg, met and rif genes on the
chromosome.
I will keep you updated about this part

27
Questions to answer in your Report on Experiment II:
Mutations in Bacteria
1. You plated 1ml of an overnight culture (O/N) of HR171 on
your LB rifampin plate. A typical O/N
contains about 5 x 109 bacteria/ml. After incubation how many
colonies did you observe?
2. What was the apparent frequency of rifampin resistant
mutants in the sample you plated?
3. Would you expect the fraction of bacteria that are
spontaneous rifampin resistant mutants to be higher
or lower in a culture you’ve propagated many times or one you
had just started from a single colony of
sensitive bacteria? Why?
4. What would you expect to be more frequent in a culture you
have propagated, Met- mutants or rifampin
resistant mutants? Why? Note: The culture was grown in
media with methionine and without
rifampin.
Extra credit:
5. Rifampin inhibits the β subunit of the E. coli RNA
polymerase. A typical rifR mutant contains a mutant
gene that produces a β subunit that no longer binds rifampin.
Propose one other genetic alteration by
which an E. coli might become resistant to the drug rifampin
(without mating with other bacteria) and
the molecular basis for this other resistant phenotype. What is

your evidence this other type doesn’t
exist?
Conjugation
6. In your Hfr cross, why didn't the KL16 strain grow on the
selective plates?
7. In your cross, were there more His+ recombinants than Trp+
or Arg+ recombinants? If so, why?
8. Would you expect more of the Arg+ recombinants to be
rifampin sensitive than either the His+ or Trp+
recombinants? If so, why?
9. Were any of the His+ recombinants also Trp+? Were any
Arg+? Were any rifampin sensitive? Plot
your data to see if it agrees with the expected results for where
the rifampin resistance mutation lies.
Transduction
10. You transduced your rifR HR171 with P1 grown on KL227
metA [Met-]. You selected Arg+
transductants. For the class as a whole, what percentage of
Arg+ transductants were Met-, what percent
were rifS? Can the rif and arg markers be co-transduced with
the met marker? If so what is the order of
these three genes from the combined data? Is this consistent
with which recombinant type is the rarest
from the three factor cross? How can you tell?
end

28
Experiment III: Transposon Mutagenesis and Gene Mapping
Transposable elements (also known as transposons) are small
DNA fragments that can move from one
site in DNA to another. Transposons can insert into new
locations throughout the DNA of the bacterial cell in
which they are present. Some transposable elements can
integrate almost anywhere in the genome including
into the middle of any pre-existing gene. When this happens the
transposon almost always inactivates the gene
thereby creating a mutant. Of course, if the gene is an essential
one (and since genes are haploid in bacterial
cells) this will kill the bacterium, and we will never be able to
isolate this particular mutant. Often, however,
the integration will be in a non-essential gene whose mutant
phenotype can be detected.
Transposon-generated mutations are especially useful because
the transposon then marks the mutant
gene both genetically and physically. It marks the mutant
genetically if the transposon contains a detectable
gene. The transposon-generated mutant gene can then be
followed by mapping the location of that gene. The
transposon marks the gene physically because the DNA of the
transposon then exists as a several thousand base
pair insertion into the DNA of the mutated gene. The size and
the nucleotide sequence of the transposon DNA
are already known for many transposons. A partial sequence of
the gene in which the transposon has inserted
can, therefore, be determined by sequencing from a primer

complementary to a sequence near the end of the
transposon.
To isolate bacteria in which a transposon has inserted into the
chromosome the transposon is introduced
into the cell on a DNA that cannot replicate in that cell (a
“suicide vector”). We shall use as a suicide vector, a
plasmid (pSAM_DKm) that contains the origin of replication of
an E. coli plasmid R6K, but not its replication
protein called π (pi), encoded by the pir gene. This plasmid can
replicate only in bacterial cells that were
engineered to carry the pir gene. If pSAM_DKm plasmid is
introduced into a cell that does not carry the pir
gene the only way the cell can become resistant to kanamicin
(carried by the transposon) is if the transposon is
transferred into another self-replicated DNA molecule in the
cell (another replicon), either the chromosome or a
plasmid.
In this experiment, you will insert the Mariner-Km transposon
into the chromosome of the P. putida
strain 2440 and screen for mutations that will inactivate the
ability of this strain to use levulinic acid as the
carbon and energy source. To date these genes are unknown.
Indications have been obtained pointing to the
existence of several such genes. The discovery of all such
genes will help to understand how P. putida
manages to use this unusual carbon source in contrast to many
other bacteria.
29

30
Experiment III: Day 1 (10/13) Purification of Plasmid
Containing the HiMar-Km Transposon
A culture of the strain CB2870 carrying the plasmid
pSAM_DKm which contains the HiMar-Km transposon
will be ready for you. Use it to purify the plasmid by the
following method:
Qiagen DNA Prep Protocols for 4.5 ml Cultures
1. Overnight cultures of 5ml in LB medium + Km + Amp + DAP
(0.3mM).
2. Centrifuge three 1.5 ml aliquots in 2ml microfuge tubes for
30s -1 min at maximum speed.
3. Discard the supernatant carefully (keep the pellets) and
resuspend each pellet in 0.5 ml STE buffer.
4. Combine the suspensions in one microfuge tube
5. Centrifuge as in step 2 and carefully discard the supernatant
(make sure that pellet does not come loose)
6. Suspend pellet in 250µL Buffer P1 (kept cold).
7. Add 250µL Buffer P2 (Lysis Buffer). Mix by inversion 10
times. Do not vortex!
Do not exceed 3 min of the lysis.
8. Add 350µL Buffer N3. Mix well by inversion as above. Do
not vortex.
9. Incubate on ice 5 min.

10. Centrifuge at maximum speed for 10 min. Team up for the
use of the centrifuges.
Supernatant should be clear.
11. Transfer the supernatant onto the Spin Column.
12. Centrifuge at 8,000 x g for 1 minute.
13. Discard the flow through.
14. Wash column with 100µL Buffer PB, centrifuge as in step
12. Discard flow through.
15. Wash column with 750µL Buffer PE, centrifuge as in step
12. Discard flow through.
16. Centrifuge the empty column for an additional 1 min at
8,000 x g.
17. Transfer column to a clean 1.5ml microfuge tube.
18. Add 50µL buffer EB to the center of the column.
19. Wait 5 min.
20. Centrifuge as in 16.
21. Save the flow through and store at 4°C.
This is your purified plasmid DNA to be used for transposon
introduction into the P. putida strain.
31
Experiment III: Day 1 Worksheet Name:
1. What happens upon addition of the Lysis Buffer (P2) to

bacterial cells?
2. Why is it so important to NOT vortex at various steps of
plasmid DNA purification?
3. Why should we not exceed 3 minutes for the lysis?
4. What happens upon addition of the Neutralization Buffer
(N3) to the lysate?
32

Experiment III: Day 2 (10/18): Concentration of DNA by
Ethanol Precipitation
1. Add 0.1 volume of 4M sodium acetate solution to your DNA
solution (for volume, see day 1, step 18).
What is a ‘volume’?
2. If available, add 1µl of “*Pellet Paint”.
*Note: This may already be added to the ethanol for you for
you. If this is the case vortex the ethanol
solution before using in point 3. since the Pellet Paint would
have precipitated.
3. Add 2 volumes of absolute ethanol. Mix well.
4. Mark the side of the tube where you expect the precipitate.
5. Centrifuge at max speed for 10min.
6. Decant the ethanol.
7. Wash the precipitate with 400µl of 70% ethanol.
8. Centrifuge as before.
9. Decant the ethanol.
10. Leave the tube opened to dry the ethanol for at least 20 min.
Note: If the SpeedVac air dryer is available, you may use it to
decrease your wait.
11. Dissolve the precipitate in 15µl of dH2O.
12. Store at 4°C.
This is your DNA of the pSAM_DKm plasmid – the donor of
the HiMar-Km transposon.
33

Experiment III: Day 2 Worksheet.
1. Why are we adding sodium acetate before ethanol
precipitation of DNA?
2. Describe what ethanol does to DNA.
3. Why must we be careful to allow all the ethanol to evaporate
before dissolving the DNA precipitate?
4. Why must we be sure to dissolve our precipitated product in
only water?

Name:
34
Experiment III: Day 3 (10/20): Electroporation of Pseudomonas
putida (strain 2440).
Note: Each student will prepare two tubes that are combined in
step 4. One student per bench will prepare a
second set of two tubes to serve as a control. Do not add DNA
to the control tube. Protecting sterility in the
following procedure is of utmost importance.
1. Place 1.5ml of the overnight culture of P. putida strain
KT2440 into each of two 2ml microfuge tubes.
2. Centrifuge 1 min at 7,000 x g.
3. Wash twice with 300µl 300 mM sucrose each.
Note: Make sure that the pellet is completely resuspended each
time.
4. Suspend each portion in 80µl 300mM sucrose and combine.
5. Add 100µL culture to your 15µl of dissolved pSAM_DKm
plasmid DNA (directly into cuvett)
6. One person per bench uses, in addition, 100µl of the electro-
competent culture without DNA. This is
your control.
7. Transfer to electroporation cuvette.

8. Electroporate each at 2.5kV, 25µF 200Ω in 2mm gap cuvette.
Record reading (≈ 4.7 - 4.8 ms).
9. Add 1ml LB to cuvette.
10. Pour contents into a 2 ml microfuge tube.
11. Shake for 1.5hrs at 32°C.
12. Plate 0.1ml of each onto a M9 + glucose + Km(100µg/ml)
plates.
13. Save your culture for the next class. It can be concentrated
and replated if there are too few colonies on
the first plate.
If this is the case centrifuge the suspension as in 2.
14. Decant supernatant.
15. Resuspend the pellet in the medium remaining in the tube.
16. Plate entire volume onto another M9 + glucose + Km plate.
17. The persons preparing control samples plate the
concentrated control onto one M9 + glucose + Km plate
18. Incubate the plates at 32° C for 2 days.
35
Experiment III: Day 3 Worksheet Name:
1. Why are we incubating the culture after the electroporation in
a medium that does not contain
antibiotic?

2. What is going to happen in the cell after the electroporation?
3. What is the control ‘controlling’ for?
36
Experiment III: Day 4 (10/25) Screening for Mutants
1. Examine the results of your electroporation plates. Count the
colonies.
2. Mark plates containing minimal agar + levulinic acid and
minimal agar + glucose at the top.
3. Use the papers with the grid provided to you.
4. Using a toothpick or a wire, patch colonies onto plates as
follows. Be careful to preserve sterility.
5. Repeat such that you screen at 100 - 200 colonies. In this
type of procedure the more is the better!
6. Incubate the plates at 30°C for 2 days.

Experiment III: Day 5 (10/27): Purification of Mutants
1. Identify colonies that grow on glucose, but not on levulinic
acid plates. These are, potentially, your
mutants.
2. Divide an M9-glucose and an M9 levulinik acid plate into 4
quadrants each.
3. Streak one of your mutants per quadrant. Share plates if
necessary.
Note: When you streak bacteria you should always streak for
single colony isolation.
4. Use one plate for 4 different colonies. If you don’t have a
mutant of your own find someone that has two
mutants and use one of those. Alternatively, use one of your
colleagues’ mutants, but keep track on which
one it is.
5. Number the colonies with consecutive numbers throughout
the entire class so we can identify them
after sequencing. Make note of your mutant number for your
report.
6. This assay verifies that you have indeed a mutant unable to
grow on levulinic acid.
7. Incubate plates at 32°C.
Experiment III: Day 6 (11/1): Culturing of the Transposon
Mutants

1. Identify a well-isolate colony of the mutant unable to grow
on levulinic acid.
2. Inoculate 4ml LB.
3. Incubate at 32°C overnight.
Note: it is CRITICAL that you use an isolated colony and that
you record the mutant number(s).
37
Experiment III: Day 7 (11/3): Isolation of chromosomal DNA
(Promega Kit)
1. Centrifuge 1 ml of overnight culture at 13,000–16,000 × g,
for 1 min (use 1.5ml microfuge tube).
Discard the supernatant (repeat if small cell pellet).
2. Add 600µl Nuclei Lysis
Solution
. Pipet gently to mix.
3. Incubate for 5 minutes at 70°C, then cool to room

temperature.
4. Add 3µl of RNase

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