Protocols Experimental Techniques of Molecular Biology and Protein Expression Summer term 2017 Cloning and expression of recombinant proteins in E. coli for the development of a multifunctional membrane for water purification Student project MultiBrane Prof. Dr. Budisa Supervisor: Saba Nojoumi, Franz-Josef Schmitt
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Protocols
Experimental Techniques of Molecular
Biology and Protein Expression
Summer term 2017
Cloning and expression of recombinant proteins in E. coli for the development of a
multifunctional membrane for water purification
Student project MultiBrane
Prof. Dr. Budisa
Supervisor: Saba Nojoumi, Franz-Josef Schmitt
1
Microbial cultivations
In this project we only worked with the coliform bacterium Escherichia coli. E. coli is one of
the best studied microorganisms and is commonly used in molecular biology. Naturally, E. coli
occurs in the intestine of mammals as an important part of the gut microbiome. Regularly, E.
coli laboratory strains are regarded as S1 organism, meaning that they are non-pathogenic
and require no special safety measures. Nevertheless, there are also harmful, toxin-producing
E. coli strains and as a scientist working with genetically modified organism we always took
care of an appropriate and sterile working manner.
Figure 1| E. coli cell schematic, source: Aqua di Cutinase project TU Berlin
2
For the cultivation of our E. coli strains we normally used LB medium (liquid broth medium)
that was supplemented with Kanamycin (50 mg/ml). Kanamycin is an antibiotic that is used to
select microorganism. We selected E. coli cells which had uptaken our plasmid and therefore
gathered a kanamycin resistance.
Table 1| LB medium recipe
component [g/L]
Trypton or Pepton 10
Yeast extract 5
NaCl 10
Kanamycin 0.05
dest. water
(adjust pH 7)
add to the desired volume
Agar (for solid media only) 1,5%
Liquid medium was prepared for cultivations of E. coli for transformations or protein
expression. Such cultivations were carried out in small cultivation tubes (10 ml) or in shake
flasks up to 1 L, which were incubated on a shaker platform at 200 rpm and 37 °C for regular
cultivations but 30 °C for protein expression.
Solid medium was prepared in agar plates to streak out E. coli or plating cells
subsequently after transformation to have the ability to select single clones. Incubation was
performed at 37 °C in a static incubator.
For all cultivations, it was taken care to take sterile materials and also work sterile, whenever
possible such work was done under a sterile bench.
3
OD measurement
To monitor growth and culture density we used OD600 measurements. Thereby, the density of
a cell suspension is determined at 600 nm in a spectrometer with cell-free medium as a
reference. The result is an absorbance value that should never exceed 1. If so, the measured
solution must be diluted. We usually measured in a 1 ml cuvette.
4
Competent cells
For the transformation of foreign DNA to E. coli the cells need the ability to take this DNA up.
Such cells are called competent and they need special treatment to increase their membrane
permeability for the uptake.
We applied two different protocols to generate competent cells:
● CaCl2 competent cells (chemically competent)
A 4 ml overnight culture of E. coli cells is used to inoculate 400 ml of fresh LB medium. The
cells are subsequently incubated until an OD600 of maximal 0.7 is reached. The culture is
portioned then into 50 ml reaction tubes and cooled on ice. Next the cells are harvested by
centrifugation at 4 °C for 10 min at a speed of 5000 x g. Cell pellets are cooled on ice and
resuspended in 10 ml of precooled MgCl2 solution (100 mM). The resuspension is chilled on
ice for approx. 30 minutes. Thereafter two aliquots are unified and centrifuged at 4 °C for 10
min at a speed of 4000 rpm. The pellets are resuspended in 2 mL sterile ice-cold CaCl2 solution
(100 mM CaCl2, 15% glycerol) each. The cells are now assumed to be competent.
Competent cells are aliquoted in portions of 50 or 100 μL, frozen in liquid nitrogen and stored
at -80 °C.
● electrocompetent cells
A 2-4 ml overnight culture of E. coli cells is used to inoculate 200 ml of fresh LB medium.
The culture is incubated at 37 °C and 200 rpm until the OD600 reaches 0.3 - 0.5. The cells are
subsequently harvested by centrifugation for 5 min at 4 °C and 3000 x g). The pellet is washed
twice with ice-cold 10% glycerol and afterwards resuspended in ice-cold 10% glycerol to a
final OD600 around 60 – 70. This cell solution is aliquoted in portions of 50 -100 µl, frozen in
liquid nitrogen and stored at -80 °C.
5
DNA amplification with Polymerase chain reaction (PCR)
Polymerase chain reaction, short PCR, has become the common procedure to amplify DNA
in molecular biology. All needed is a DNA template, which contains the desired DNA
sequence, primer - small DNA oligos that match the beginning and the end of the desired DNA
sequence perfectly and a DNA polymerase - an enzyme that can produce copies of DNA.
There are basically 5 steps:
● STEP 1: initial denaturation of the template DNA - to get single strand DNA (otherwise
the DNA polymerase cannot copy the DNA)
● STEP 2: denaturation of the template DNA
● STEP 3: Annealing of the primer
● STEP 4: extension of the primer along the DNA template (DNA copying by DNA
polymerase)
● STEP 5: final elongation - DNA polymerase finishes the copying process for all
templates
These steps are performed at specific temperatures and step 2 - 4 are repeated for several
times which leads to n2 -DNA fragments in each cycle. Optional the reaction can be stopped
and stored at 4 °C for a longer time period in the end.
Table 2| PCR programm
Step Temperature
[°C]
Time [min] Cycle nb.
1 Initial denaturation 95 3:00 1
2 Denaturation 95 0:30 30
3 Annealing 0:30 30
4 Extension 72 30
5 End elongation 72 10:00 1
6 Hold 4 infinity infinity
The annealing temperature depends on the melting temperature of the primers and the
reaction mixture components. It has to be calculated for every PCR which can be done online.
6
The timespan for the extension depends on the size of the DNA fragment and the synthesis
velocity of the applied polymerase.
We worked with the Taq-Polymerase (1 kb/min) and the Q5® High-Fidelity DNA Polymerase
by NEB (2 kb/min). the difference between both polymerases is the synthesis accuracy, which
is higher for the Q5®. Therefore, control PCRs were performed with Taq DNA Polymerase and
amplification of genes for further cloning was performed with Q5® DNA Polymerase.
We prepared a PCR mix freshly for every amplification after the following scheme:
Table 3| PCR mix pipetting scheme
amount comments
DNA template < 1 ng DNA amount needs to be
calculated
polymerase buffer
(10x)
2 µl specific for each polymerase
polymerase 0.125 - 0.25 µl
depends on the polymerase
protocol
primer #1 (10 µM) 0.5 - 1.25 µl
primer #2 (10 µM) 0.5 - 1.25 µl
dNTPs (10 mM) 0.5 µl
nuclease-free water up to 25 µl
Note: The primers were ordered from Sigma-Aldrich after in silico design. After arrival, they
were diluted to 10 µM aliquots and stored at -20 °C.
The result of the PCR is evaluated via gel electrophoresis.
7
Gel electrophoresis
To separate and analyse DNA fragments we used 1% agarose gels. Agarose was boiled with
the running buffer (50x TAE buffer: Tris, acetic acid, 0.5M EDTA) until the solution becomes
completely clear. This solution can be stored at 60 °C. Each gel is prepared with an appropriate
comb and a staining reagent is added. For staining we used either GelRedTM or ethidium
bromide and to determine the DNA fragment size 8 µl of DNA ladder (1 kb, GeneRulerTM
Thermo Fisher). The samples were diluted with nuclease-free water and loading buffer (6x,
Thermo Fisher) to a final volume of 6 µl and loaded to the gel. To run the gel electrophoresis
voltage is applied: we regularly applied 90 V for a 1 h run. However, sufficient separation must
be assured. There are loading buffer systems that include pre-stained DNA fragments (100
bp) which can be monitored during the run for their movement.
The run is stopped by disconnecting the voltage and transferring the gel to a photo-
documentation station. This instrument is used to read out DNA gels as it illuminates the
stained DNA. GelRedTM and ethidium bromide are both fluorescent when exposed to
ultraviolet light. The exposure can be monitored and captured by a camera. The gel images
can then be analysed.
Note: Ethidium bromide makes DNA visible by intercalation. This is could lead to mutations
even in living organism (mutagenic). The handling of ethidium bromide therefore need special
safety measures. GelRedTM makes DNA visible by binding to the sugar-phosphate backbone
and is thus assumed to be non-mutagenic.
8
Gel extraction
To separate DNA fragments of different size and utilization of these in further cloning steps
gel extraction was conducted.
Therefore, the mix that contains the DNA is conditioned with loading buffer and loaded to the
gel. The gel in this case has much larger gel bags thus a large volume can be loaded.
The gel is prestained with either GelRedTM or ethidium bromide and to determine the DNA
fragment size 8 µl of DNA ladder (1 kb, GeneRulerTM Thermo Fisher) is loaded to a small gel
bag. To run the gel electrophoresis voltage is applied: we regularly applied 90 V for a 1 h run.
However, sufficient separation must be assured. There are loading buffer systems that include
pre-stained DNA fragments (100 bp) which can be monitored during the run for their
movement.
The run is stopped by disconnecting the voltage and transferring the gel to a photo-
documentation station. This instrument is used to read out DNA gels as it illuminates the
stained DNA. GelRedTM and ethidium bromide are both fluorescent when exposed to
ultraviolet light. The exposure can be monitored and captured by a camera. The gel images
can then be analysed.
The exposure of DNA by ultraviolet light induces DNA damage. Since we want to utilize the
DNA from the gel afterwards, the exposure should be short with a low UV light intensity.
Furthermore, this also protects the person working directly at the illumination desk. This
person has to wear UV-protecting glasses and skin protection. The desired DNA fragment is
then cut out of the illuminated gel with a scalpel and transferred to a 1.5 ml reaction tube.
Depending on the applied kit, the reaction tube must be weighed without and with gel pieces
in to determine the weight of these. We used two different Gel extraction kits: Roti®-Prep Gel
Extraction, Roth and GeneJET Gel Extraction Kit, Thermo Scientific. The gel extraction was
performed according to the manufacturers manual. The eluted DNA concentration was
determined by UV - spectroscopy.
Note: Ethidium bromide makes DNA visible by intercalation. This is could lead to mutations
even in living organism (mutagenic). The handling of ethidium bromide therefore need special
safety measures. GelRedTM makes DNA visible by binding to the sugar-phosphate backbone
and is thus assumed to be non-mutagenic.
9
DNA concentration measurement
DNA concentration was measured after each step of our cloning protocol. The DNA solution
is measured with a UV-spectrometer in reference to an appropriate buffer blank. In order to
detect protein impurifications and solvent residues, the measurement includes four
wavelengths.
260 nm - DNA
280 nm - proteins
230 nm - organic impurifications
340 nm - ethanol
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DNA purification
After enzymatic treatment of DNA with either polymerases or restriction enzymes the DNA
needs to be purified from theses enzymes and their buffers. Regularly, we used DNA
purification Kits for that (GeneJET PCR Purification Kit, Thermo Scientific). The purification
was performed according to the manufacturer’s manual.
In case we wanted to monitor the DNA between cloning steps we did a gel purification or gel
extraction.
11
Restriction digest
Restriction enzymes are part of the bacterial immune system and cut DNA specifically. For
this these enzymes need a specific recognition sequence called restriction site. Restriction
sites are mostly palindromic DNA sequences. When such as sequences is cut by a specific
enzyme this can either result in blunt ends without overhangs or sticky ends with overhang.
The enzymes that we applied all create a 5`prime overhang.
To clone our gene of interest we need to cut open the plasmid backbone and make the insert
fit into that frame. For each cloning procedure, we apply two different restriction enzymes to
generate overhangs with different base pairs. Thus, the plasmid cannot repair itself (auto-
ligation).
We prepared a digestion mix freshly for every restriction digest after the following scheme on