Download Exercise 8

BC2004
Bacterial Transformation
Exercise 8
Spring Semester 2005
weeks of 3/28-4/1 and 4/4-4/8/05
Transformation of bacteria is the process in which the cell takes up a molecule of DNA from the
environment and incorporates at least some its information into its own heredity. The DNA may
contain information that improves the ability of the bacterium to survive and multiply in a given
environment, and some bacteria are naturally competent for transformation. Natural transformation
is one mechanism by which bacteria transfer genes between contemporaries, allowing the phenomenon
of “lateral gene transfer;” this is thought to be a major mechanism by which antibiotic resistance has
become widespread among many different types of bacteria. Genes carried on relatively small circular
DNA molecules known as plasmids seem particularly suitable for this mechanism of genetic
communication among bacteria.
However, the foreign DNA may contain information that is incompatible with normal activities of the
cell and thereby be hazardous to the health and reproduction of the bacterium. All bacteria that have
been tested contain enzymes, called “restriction endonucleases,” that attack and digest foreign DNA
(primarily from viruses) that enters the cell, thereby eliminating the foreign information (and
incidentally providing a nucleotides snack for the cell). Nevertheless, several types of bacteria can be
gently abused (by exposure to chemicals, heat or electrical pulses) in such a way that they will accept
and replicate foreign DNA. Such practices have become a major tool in molecular genetics, where
moving genes from molecule to molecule of DNA and transforming suitably cooperative (or coerced)
bacteria with “engineered” DNA allows the chemical and biological analysis of those molecules.
In this exercise, you will attempt to transform ampicillin-susceptible cells of the bacterium Escherichia
coli with an engineered plasmid pBLU. The plasmid normally carries an ampicillin resistance gene
(ampR) that confers resistance to the antibiotic ampicillin and a gene (lacZ) that guides the synthesis of
the enzyme β-galactosidase. In the DNA preparation you will use, some of the plasmid molecules will
be this version of the plasmid, but there will also be some plasmid molecules that have been engineered
to contain a third gene (as a molecular detective in the lab, you will be trying to sort out the versions of
the plasmid that are contained within your bacteria). The unknown gene has been inserted into the
plasmid within the lacZ gene, thereby interrupting the lacZ gene and preventing the expression of βgalactosidase. In this molecular arrangement, the normal plasmid is regarded as the vector (the carrier,
the transmitter) of the inserted gene, and the modified plasmid is a recombinant DNA molecule
because it contains DNA from two sources combined in one molecule.
You will gently abuse the E. coli cells (in the presence of exogenous DNA) by soaking them in a
calcium chloride solution (CaCl2) at 0oC for several minutes and then heating them briefly at 42oC.
After a short recovery period, during which transformed cells will begin to express their newly
acquired genes, the cells will be plated on two media: Luria-Bertani (LB) agar, and LB agar
supplemented with ampicillin and “X-gal” (5-bromo-4-chloro-3-indolyl galactoside). All the viable
(living) cells will grow and reproduce on LB agar plates, whether or not they have been transformed.
Only transformed cells (“transformants”) will be able to grow on the medium containing ampicillin.
BIOL BC2004, Spring 2005, Lab exercise 8-1
Figure 1. pBLU plasmid map showing locations of amp R gene, lacZ gene, origin of replication (ORI), and restriction
recognition sites (we’ll talk more about these in lab exercise 10). Diagram is from:
“http://www.dnalc.org/resources/NucleotideSeqs.html”.
X-gal is a substrate that is hydrolyzed by β-galactosidase to yield a blue product. Cells transformed by
the vector only will express the lacZ gene, attack the X-gal, and form blue colonies; cells transformed
by the recombinant molecule will not express the (interrupted) lacZ gene, will not attack the X-gal, and
will form white colonies.
In preparation for lab, complete the following diagram:
A. control E.coli on LB plates
predicted colonies : relative number and color
B. control E.coli on ( LB + amp + X-gal) plates
predicted colonies : relative number and color
Have these cells be transformed successfully?
Have these cells be transformed successfully?
If so, with what form(s) of the plasmid?
If so, with what form(s) of the plasmid?
BIOL BC2004, Spring 2005, Lab exercise 8-2
C. plasmid-transformed E.coli
on LB plates
D. plasmid-transformed E.coli
on (LB + amp + X-gal) plates
predicted colonies : relative number and color
predicted colonies : relative number and color(s)
Have these cells be transformed successfully?
Have these cells be transformed successfully?
If so, with what form(s) of the plasmid?
If so, with what form(s) of the plasmid?
Report
(although you will work in pairs, each student will independently write
and submit a formal lab report, due at the beginning of lab next week)
A report will be completed for this exercise that will include your predictions/hypotheses, your data,
your calculations of the proportions of transformants of each type, and a discussion of your data.
Explain the bases for your predictions in your introduction, what you did (with appropriate citations
and in past tense) in your materials and methods, your data in table and written forms in your results
section, and an explanation of what happened on each of your six Petri dishes. Also include a
discussion of the significance of any colonies observed on the (LB + amp + X-Gal) plate spread with
a sample of bacterial cells that were not exposed to the plasmid DNA. What would be the source of
such colonies (aside from flawed aseptic technique)? If those clones arose by some mechanism other
than transformation, how could they be distinguished from the transformants? [This is not an easy
question; think molecularly.]
If you failed to obtain transformants, suggest a way(s) in which the procedure could be modified to
increase the likelihood that transformants were not overlooked. If you succeeded in obtaining
transformants, suggest a way(s) to increase the number of transformant clones yielded by the
procedure.
You should follow the DETAILED instructions on how to write a lab report that are given in Chapter
8 of A Short Guide To Writing About Biology. Your report will be graded according to the
standards outlined in this book.
BIOL BC2004, Spring 2005, Lab exercise 8-3
Procedure (work in pairs)
A. First week: transformation.
1.
Obtain two small sterile microfuge tubes. Label one tube “C” (for no plasmid DNA
control) and one tube “T” (for transformation mixture).
2.
Aseptically transfer 0.5 ml of ice-cold CaCl2 in the “C” tube and 10 μl of plasmid DNA
in the “T” tube. Keep both tubes on ice.
3.
Suspension: Using a sterile inoculating loop, transfer E. coli cells from colonies (try to
scoop up bacteria from a section that is about the same diameter as a pencil eraser—do
not scoop up ANY agar!) on LB agar to the “C” tube, using sufficient cells to make a
turbid (i.e. cloudy) suspension. Vigorously wiggle the loop in the calcium chloride
solution and scrape it repeatedly against the side of the tube to dislodge the bacteria from
the loop. Vortex the suspension to disperse the cells evenly; it is important that the cells
are completely suspended and are not clumped. Return the tube to the ice to chill the cell
suspension.
4.
Transformation: At “OT,” (zero time) transfer 0.25 ml of the cell suspension to the “T”
tube containing the DNA, mix well by vortexing briefly. Allow both tubes to remain in
the ice for 15 minutes. [During this waiting period and the waiting period in step 6, you
may prepare dilution tubes and plates for step 7. It might also be wise to review (before
lab) the procedure for spreading bacteria for viable cell counting from Exercise 4.]
5.
Heat shocking: After the 15-min. ice-cold incubation, transfer the tubes to the 42oCwater bath for 90 seconds (the timing is important!). Gently agitate the tubes two or
three times while they are in the water bath. Return the tubes to the ice for 2 minutes.
6.
Recovery: Aseptically add 0.25 ml LB broth to each tube and mix each tube gently as
you transfer it to a rack at room temperature. Let the tubes stand at room temperature for
10 minutes.
7.
Plating:
a. Obtain three tubes containing 0.990 ml of LB broth per tube. Label them “T 10-2,” “T 10-4"
and “T 10-6."
b. Obtain three plates containing LB medium and three plates containing (LB+amp+X-gal)
medium.
Label the plates appropriately, including your initials and lab section number on each
plate.
1 plate of - LB “C”
1 plate of - LB + amp + X-gal “C”
1 plate of- LB “T 10-4"
1 plate of - LB “T 10-6”
2 plates of - LB + amp + X-gal “T”
BIOL BC2004, Spring 2005, Lab exercise 8-4
c. When the 10-minute recovery period is complete, mix the “T” suspension well and dilute as
follows.
transfer 10 μl of suspension to the T 10-2 tube, mix well.
transfer 10 μl of the T 10-2 dilution to the T 10-4 tube, mix well.
transfer 10 μl of the T 10-4 dilution to the T 10-6 tube, mix well.
You now have serial dilutions of your transformation mixture. Note that you won’t be
using the T 10-2 tube any further.
d. Follow the directions for spread-plating of bacteria for viable cell counting that you used in
Exercise 4, and spread onto your labeled plates:
100 μl of undiluted “C” suspension onto one plate of LB
100 μl of undiluted “C” suspension onto one plate of LB + amp + X-gal
50 μl if “T 10-4" onto one plate of LB
50 μl if “T10-6" onto one plate of LB
100 μl of undiluted “T” suspension onto each of two plates of LB + amp + X-gal
e. Give your plates to your instructor for overnight incubation at 37º C. Following overnight
incubation, your plates will be wrapped with Parafilm (they need O2 to grow in the incubator
overnight), and placed in the fridge until your lab section next week.
B. Second week: observations and calculations
1.
Observe the plates that were spread with the cell suspension that was incubated without
added DNA (the “C” plates). The control plate of LB should contain a solid mass (a
lawn) of bacterial growth, but the control plate of (LB+amp+X-gal) should not have any
colonies. If there are colonies, count them and record the number in the data table.
If colonies have developed on this plate, where did they obtain the information to resist
the antibiotic?
2.
Observe the LB plates that were spread with dilutions of the transformation mixture (the
“T” plates). Count the number of colonies per plate and, using the counts on the plate
(T 10-4 or T 10-6) on which the mean number of colonies is closer to 100, and calculate
the total cell density in the transformation mixture (see Exercise 4):
mean number of colonies/plate x 1/D.F. = no. cfu/ml transformation mixture
volume of dilution per plate
BIOL BC2004, Spring 2005, Lab exercise 8-5
3.
Observe the (LB + amp + X-gal) plate that was spread with undiluted transformation
mixture. (As a side note, consider why you plated dilutions of untransformed cells, but
plated undiluted transformed cells.)
These colonies presumably arose from transformants. Count the blue colonies and the
white colonies separately and record the counts in the data table. Calculate the number of
cfu of each type that formed in the transformation mixture.
4.
Calculate the proportion of the cell population accounted for by transformants that
received the vector only, and the proportion accounted for by transformants that received
the recombinant molecule (those that contained the third gene that interrupted the Lac Z
gene.
Data Table. Count and record the number of colonies per plate. Use these data to
calculate the cfu per mL of culture. Note that you won’t fill in the grey boxes.
Population
plated
Plating
medium
ml/dilution
no. colonies/plate
blue
Control, No
plasmid DNA
Transformation mixture
blue
white (LB:
total only)
--
should be
a” lawn”
LB
0.1 ml
undiluted
LB+amp
+X-gal
0.1 ml
undiluted
LB
0.05 ml/10-4
--
(1)
--
(2)
LB
0.05 ml/10-6
--
(1)
--
(2)
LB+amp
+X-gal
(1)
white (LB:
total only)
cfu/ml
--
0.1 ml
undiluted
(2 plates)
Use whichever of these numbers is closest to “100” to calculate your total cfu/mL = (2).
BIOL BC2004, Spring 2005, Lab exercise 8-6
Calculations: Use the cfu/mL (2) that you calculated on the previous page from the (1)
number that is closest to 100 for your total cfu/mL
1.
proportion of the recipient population accounted for by ampicillin-resistant transformants
that received vector only (the blue cfu on LB+amp+X-gal):
blue cfu/ml ÷ total cfu/ml = _____________________
2.
proportion of the recipient population accounted for by ampicillin-resistant transformants
that received the recombinant molecule (the white cfu on LB+amp+X-gal):
white cfu/ml
3.
÷
total cfu/ml = _____________________
proportion of the recipient population accounted for by ampicillin-resistant transformants
(total of blue cfu and white cfu on LB+amp+X-gal):
(blue cfu/ml + white cfu/ml) ÷
total cfu/ml = _____________________
This is your transformant efficiency (we expect it to be very small).
BIOL BC2004, Spring 2005, Lab exercise 8-7
Materials and set-up instructions
Equipment:
per group of FOUR students:
1
vortex
per PAIR of students:
2
transformation tubes (sterile 1.5-mL Eppendorf tubes)
1
inoculating loop (sterile)
3
1.0 ml pipettes (sterile)
1
small blue pipette pump
1
micropipettor, 40-200 uL
1
micropipettor, 5-50 uL
1
rack of sterile micropippettor tips
1
tip discard beaker, labeled
1
“hockey stick” glass spreader
1
covered beaker of alcohol, labeled
1
turntable
1
Bunsen burner/striker
1
ice bucket (needed per PAIR of students)
1
Eppendorf tube rack
1
Sharpie pen
Per group of EIGHT students
1
42-degree C water bath, needs to be turned on well before lab section, with
non-mercury (preferred) thermometer
Media:
per PAIR of students:
1
blue-capped tube containing 5 ml 50mM CaCl2, labeled
1
red-capped tube containing 5 ml LB broth, labeled
3
yellow-capped tubes containing 0.990 ml LB broth, labeled
3
LB plates, labeled
3
(LB + amp + X-gal) plates, labeled
There should be extra supplies of all materials available in each lab room for each section.
BIOL BC2004, Spring 2005, Lab exercise 8-8