Lab 9: DNA Ligation and Transformation of E.

coli and Protein

Crystallization
Jillian Johnstone
Partner: Rachel Johnstone
Section 219
April 4, 2016
Objective:

The objective of laboratory 9 was to one, get competent E. coli cells to transform by
taking up a plasmid vector which had been modified by a DNA insert, and
subsequently, to grow these cells on an agar plate and analyze the colonies that
grew (for use later in DNA purification???). Another objective of this lab was to
evaluate protein crystallization of chicken lysozyme via the hanging drop technique.

Principle of Methods:
Objective sentence
X-ray crystallography is an advanced technique for protein visualization.
When looking at a protein under even the more powerful electron microscopes,
there is only up to a four angstrom resolution. This is not a high enough resolution
to see more than the general shape of a protein. In order to visualize the structure
of the protein with up to a 0.5 angstrom resolution, one would use x-ray
crystallography. With this highly resolved view of a protein, the shape and function
of the protein can be evaluated as well as possible uses of the protein in drug
design. However, in order to use x-ray crystallography, one must crystallize their
protein of interest. As each protein will have a unique crystal structure, or space
group, and thus diffract x-rays differently, the crystal structure of the protein will
help determine information about the protein. There are many ways to crystallize a
protein including the hanging drop technique that can be used on proteins such as
chicken lysozyme. Very high concentrations of protein are added to a coverslip
which is hung above a precipitant solution that is placed into a reservoir. This
precipitant solution can contain glycol or NaCl and buffer. The coverslip is then
sealed to the well with grease. The liquid from the protein solution will precipitate
out into the solution below and a crystal will form. This procedure is done at
different temperatures as the timing of this precipitation is essential for crystal
formation (Lab Manual).
Recombinant DNA technology is extremely useful in research. By combining DNA
sequences that are not normally found together into one system, researchers are
able to analyze genes and produce desired protein products, among many other
applications. Vectors carry the gene of interest and are placed into a host cell,
which is commonly a bacteria. Bacteria take up vectors called plasmids in a process
called transformation. DNA that is too long for a plasmid vector can be placed in a
bacteriophage, cosmid, chloroplast DNA, etc., and are incorporated into
corresponding cells. In order for a bacteria to willingly take up a plasmid, the cell

must be made competent. This is normally done by placing the bacteria in cold
calcium chloride solution and then heat shocking the bacteria at 42C (Lab Manual).
The vector that is taken up by the bacteria will hopefully contain the gene of
interest. However, this will not always be the case. In order to be used in DNA
recombination, a vector must have an origin of replication, selectable marker, and
multiple cloning sites where the gene of interest can be spliced into the plasmid.
The multiple cloning sites in a vector are cut by restriction enzymes in order to allow
for the insertion of a DNA fragment, which has been cut with the same restriction
enzymes so as to match up correctly with the plasmid DNA. In order to correctly
orient the DNA in the vector, two restriction enzymes can be used, which will force
the DNA fragment to be inserted only one way. If the restriction enzymes do not
correctly cut the plasmid, or if the plasmid reconnects to itself without including the
new DNA fragment into its sequence, the bacteria will take up a plasmid that does
not include the protein of interest. When restriction enzymes are not used, a
process called TA cloning can introduce the gene of interest into the plasmid. In TA
cloning, a linear plasmid that has T overhangs is combined with a piece of DNA that
has been grown using TAQ polymerase that puts A overhangs on the ends of the
DNA fragment. The T and A overhangs will anneal together to fuse the plasma DNA
and the new DNA sequence. The plasmid will need a selectable marker. This
marker, usually for antibacterial resistance, is added to the vector so that, when the
vector is taken up by the bacteria, the bacteria will form a colony on a plate
containing antibiotics. Therefore, the only colonies that grow will contain the
plasmid unless there was an error with the antibiotic rendering it inert or there is a
mutation for antibiotic resistance in the bacteria. For example, if a plasmid has a
vector containing an ampicillin resistance gene, only transformed bacteria
containing the plasmid will grow on an ampicillin plate. However, this does not tell
one if the vector contains the DNA insert (Lab Manual).
In order to detect if the vector contains the DNA insert, a blue and white screening
test is run. When an insert is introduced to a plasmid, it can be inserted into the
middle of a LAC-Z gene, disrupting its function. When the LAC-Z gene is fully
functional, it codes for -galactosidase, a protein that cleaves lactose and lactose
derivitives such as X-Gal. When X-Gal is cleaved, its products are oxideized and
turn the colony blue, indicating that there was no insert in the vector that was taken
up by the bacteria that started that colony as its LAC-Z gene was fully functional. If
the DNA is inserted into the Lac-Z gene, no -galactosidase is produced and X-Gal is
not cleaved, leaving the colony a white color (Lab Manual).
In order to tell if the growth of bacteria on a plate is a result of bacteria
transformation or that blue and white colonies truly indicate that the insert was or
was not present in the plasmid, controls must be used. The first control is a positive
control using bacteria that definitely contain the vector but no DNA insert, as well as
a selectable marker, such as ampicillin resistance. Blue colonies will grow on an
ampicillin plate indicating that the bacteria with the insert do grow on the ampicillin
media and correctly cleave X-Gal as they contain a working LAC-Z gene. The next
control is a negative control. This control contains competent bacterial cells, usually
E. coli, which do not contain any vector. There should be no growth on an ampicillin
plate for these cells as they have no plasmid with selectable marker and thus no
anti-bacterial resistance. This indicated whether the ampicillin used in the agar

plate is working. The last marker is a positive control that uses competent cells on
a plate with no antibacterial agent. This control is used to make sure that the
bacteria cells are alive and would grow (Lab Manual).

Materials:
Crystallization buffers:
3 M NaCl
1 M NaAc (Sodium Acetate)
Chicken lysozyme- 50 mg/ml
24 well crystallization tray
dH2O
Cover slips
Grease sealant
pGEM-T Easy Vector
Insert
T4 DNA ligase
2X ligation buffer
SOC Media
LB Agar plates
Ampicillin- 50 mg/ml
100 mM IPTG NAME
5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal)
Competent cells- TOP10 or XL10-Gold
Microcentrifuge tube
Eppendorf Research Plus Micropipette- p10, p200, p1000 with corresponding tips
Spreader
Incubator
Round-bottom Falcon tubes
Water bath (42C)

Results:
Figure 1:

Figure 2:

Table 9-2: Results of E. coli Transformation

Plate

Colonies
Present?
(Yes/No)

Colony
Color(s)
(Blue/White)
Blue and
White
Blue (few
white
colonies)

Expected
Color
(Blue/White)
Blue and
White

Ligation

Yes

pGEM-T (Positive Control)

Yes

Competent Cells (Negative Control)

No

--------

--------

Competent Cells (Positive Control)

Yes

White

White

Blue

Figure A

Figure B

Figure C

Figure D

Discussion:
OBJECTIVE SENTENCE
Protein Crystallization:
None of the three wells prepared by the student in laboratory 8 showed
protein crystallization. Protein crystallization was not seen in any wells for students
in section 219. However, crystals were produced by wells prepared in Wednesday

labs and in the Thursday morning lab. No data was collected for the wells of
students in section 219. The lack of protein crystallization could have occurred for a
number of reasons. Firstly, the atmospheric conditions in the room may not have
been ideal for protein crystallization. The humidity and temperature in the room
could have changed between the Thursday morning and Thursday afternoon lab and
disrupted the crystallization of the protein.
Figure 1 above shows a sample of a well with incorrect protein crystallization.
The image shows a well with proteins that were dehydrated too quickly as indicated
by the thickened edges of the blob and the scattered look of the crystals in the
well. Figure 2 shows a sample of what protein crystallization is ideally supposed to
look like. The crystals have defined shapes, or space groups, and are distinct from
one another in the sample. These crystals can be used in an x-ray procedure.
Recombinant DNA:
MAKE SURE to include what was on each plate and why, and what was expected
and why.
One objective of laboratory 9 was to induce transformation in E. coli.
These transformed E. coli were induced via heat shock technique to take up
a plasmid that contained a DNA insert. Once the DNA were transformed, the
E. coli was grown on an agar plate and evaluated. The agar plate and broth
that was added along with the E. coli cells, contained ampicillin, IPTG, and XGal. The plasmid that was inserted into the E. coli contained a selectable
marker for ampicillin. If ampicillin was on the plate, only E. coli that took up
the plasmid would grow. However, this does not indicate whether the
bacteria took up plasmid that has a DNA insert. The IPTG induced the
production of the genes on the plasmid. X-Gal is a lactose derivative whose
products, when cleaved, oxidize and turn blue. The DNA insert was inserted
into the Lac-Z gene of the plasmid vector. If the DNA insert was correctly
taken up by the plasmid and transformed into the bacteria, this gene would
be disrupted. The -galactosidase coded for by Lac-Z would not be produced
and X-Gal would not be cleaved. Therefore, the colonies would still grow on
an ampicillin plate due to the ampicillin resistant gene on the plasmid, but
the colonies would be white. If the plasmid did not contain the insert, the
Lac-Z gene would prioduce -galactosidase which would cleave X-Gal. The
colonies would grow blue.
The ligation plate in Figure A shows the results of the transformed
bacteria. It was expected that some of the colonies would be blue, a result
of the plasmid being taken up by the bacteria but having no insert, and most
of the colonies would be white. White colonies, as aforementioned, would
indicate that the bacteria took up a plasmid that contained the DNA insert
that disrupted the Lac-Z gene. The students plate was consistent with the
expected results. There were some blue colonies on the plate. However,
most of the colonies were white. One would assume that the white colonies
contained the plasmid with the DNA insert and that the blue colonies
contained the plasmid without an insert only after comparing the results of

this plate with control groups. Control groups account for various errors that
could have occurred throughout the plating process.
The Pgem-T Easy positive control (Figure B) represented E. coli that
were induced to transform by taking up a plasmid without any introduction of
a DNA insert. The expected results of this positive control would be for there
to be growth of only blue colonies on the plate, indicating that the bacteria
successfully took up a plasmid without any insert. The students pGEM-T
Easy positive control did grow mostly blue colonies with a few cloudy white
colonies. The cloudy white colonies were attributed to contamination of
either the equipment or the plate directly throughout the course of the
procedure with a bacteria that was not ecoli or E. coli that contained a
plasmid with a DNA insert. This positive control was created to test that the
plasmid could be properly taken into the bacteria and that the Lac-Z properly
codes for -galactosidase and cleaves X-Gal. Failure of growth on this plate
could indicate that there was an issue with the bacteria taking up the vector,
the coding in the vector, or the X-Gal.
The competent cells negative control (Figure C) tested whether E. Coli
cells that did not contain the marker, and thus the selectable marker, could
grow on an ampicillin plate. There was no growth expected on this plate.
The students findings were consistent with these expectations as there was
no growth on the plate. If there was growth on this ampicillin plate, it may
indicate that there was a mutation in the E. coli batch being used where they
developed ampicillin resistance without the introduction of the vector, or that
the ampicillin used to make the plates somehow was denatured and not
functional. If this were to happen, the results of the ligation plate would be
ambiguous.
The E. coli of the competent cells positive control (Figure D) were
grown on a agar plate that did not include ampicillin. This plate showed
whether the E. coli themselves grew under normal conditions, indicating
whether the batch was viable and able to be used in further experiments.
The colonies on the competent cells positive control plate were expected to
be white, which was consistent with the garnered results. In this case, a
cloudy white is the expected color of an E. coli colony grow on basic media.
Sources: Borgon, RA and Verity, N. (2012). Quantitative Biological Methods.
Pearson.