MICB 323 2022W

Dr. Marcia Graves

MICB 323 – MOLECULAR IMMUNOLOGY & VIROLOGY LABORATORY

Project 1 (P1): Technical development

In this first lab we are mainly practicing aseptic technique while working with bacterial cultures. We are performing a
bacterial transformation to propagate a plasmid that is used for expression in mammalian cells. As part of our overall
research objectives, this plasmid would be a useful tool to visualize changes to the actin cytoskeleton in response to LPS in
cultured macrophages. Instead, we will use a staining technique (labeling cells with fluorescently conjugated phalloidin) to
address this in a future lab session.

LABORATORY 1 OBJECTIVES
Experiment #: P1-01
• To prepare chemically competent E. coli bacterial cells
• To transform recombinant plasmid DNA (LifeAct-GFP) into chemically competent E. coli bacterial cells

Experiment #: P1-02
• To pick single colonies of E. coli transformed with recombinant plasmid and grow bacterial cultures
• To isolate plasmid DNA from the bacterial cultures
• To determine DNA concentration of our purified plasmid

BACKGROUND P1-01
GENE TRANSFER: TRANSFORMATION OF E. coli

Bacterial transformation involves the following steps:

1. Preparation of competent cells.
2. Transformation of competent cells.
3. Selection of transformants.

The ability of a bacterium to take up exogenous naked DNA is called “competence.” E. coli cells are not naturally
competent, but can be artificially induced to become competent through various techniques. For example, a simple
chemical procedure uses high concentrations of CaCl2, which alters the structure of the bacterial cell wall and facilitates
the entry of foreign DNA. An alternative method (electroporation) involves the use of an electric shock to permeabilize
bacterial cells and facilitate DNA entry.

The term transformation is used to describe the uptake of DNA from the environment by a bacterial cell. This phenomenon
occurs in nature between bacteria of the same species. In the lab, this term describes the procedure of introducing
recombinant plasmid DNA into competent bacterial cells as a means to easily propagate the introduced plasmid DNA, for

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protein production and various other applications. We are transforming bacterial cells to propagate a recombinant
plasmid to purify it so that we may introduce it into eukaryotic cells.

TRANSFORMATION OF E. COLI BY HEAT SHOCK

We will be using a heat shock procedure to transform our cells. In this procedure, a combination of low temperatures and
calcium ions helps DNA bind to the cell walls of E. coli, facilitating the uptake of DNA. After incubating cells with DNA at
low temperatures, uptake is then enhanced by shocking the cells briefly at high temperatures. Cells recover over time,
and begin to express the genes on the transfected DNA. Other commonly used methods are listed in Table 1.

Table 1: Methods of bacterial gene transfer.

Method Description
Heat shock A combination of low temperatures and calcium ions helps DNA bind to the cell
walls of E. coli, facilitating the uptake of DNA. After incubating cells with DNA
at low temperatures, uptake is then enhanced by shocking the cells briefly at
high temperatures. Cells recover over time, and begin to express the genes on
the transfected DNA
Electroporation An electrical pulse causes tiny pores to form on the cell membrane, allowing
DNA to enter the cell. This has been used on bacterial and mammalian cells.

Biolistics/gene gun Involves coating tiny particles of tungsten or gold with DNA and blasting the
particles into cells. Imagine spraying a target with a pellet gun. This method
penetrates cell walls.

SELECTION OF TRANSFORMANTS:

Selection can be accomplished by introducing selectable markers to the plasmid vector. Selectable markers are generally
genes that confer resistance to antibiotics or allow auxotrophic host cells to grow on minimal medium. More on this in
P1-02 below.

PROTOCOL FOR P1-01:

Prepare your own CaCl2 chemically competent cells and then use these cells to perform a transformation using the heat
shock method.

MATERIALS:

2 ml TOP10 cells grown to O.D.600nm = 0.2 (original culture)

Aliquot of sterile LB media
Sterile water in microfuge tubes (for control)
Plasmid (life-act GFP) – we will provide you with the stock concentration in the lab
100 mM chilled CaCl2
Water bath (42°C)
Chilled microfuge tubes
Shaker
Incubator (37°C)
Ice and ice bucket
2 LB-kanamycin plates (30 µg/ml kanamycin)
2 LB plates

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PROCEDURE:

Notes before you get started:

• Competent cells are quite fragile. Treat gently (no vortexing!) and keep cold on ice at all times.
• Use sterile technique! All tips, tubes, and media used in this procedure should be sterile, and care should be used to
ensure that your cells remain free of contaminants. Make sure to sterile your bench space!
• The Bunsen burner should only be used when adding media to your tubes, and when spreading your cell cultures on
agar plates.

1. Set the test tube containing the original bacterial culture (label this OC-Top10) on ice to minimize any further
growth. Chill original culture for 10 minutes.

2. Using sterile, aseptic technique, transfer 1 ml of this OC to each of 2 chilled microcentrifuge tubes. One tube is a
control and one will be for adding your plasmid DNA. Place the test tube labeled OC back on ice.

3. Centrifuge the 2 microfuge tubes at 14,000 RPM for 2 minutes. Discard (micropipette, or gently shake out) the
supernatants into the pipette discard. Re-centrifuge the 2 microfuge tubes for 15 seconds, and use a pipette to
carefully remove the residual LB from the bottom of the tube, taking care not to remove the pellet.

4. Add 50 µl (See Pre-lab Question 1) ice cold 100 mM CaCl2 to the cell pellet in each microfuge tube and gently
finger vortex or pipet up and down with a P200 to re-suspend the cells. Place both tubes on ice for 30 minutes.
While waiting – this is a good time to pre-warm your LB media to 37°C in the walk-in incubator. Use a rack to carry
tubes, and don’t forget to label!

5. To one of the microfuge tubes containing the 50 µl cell suspension, add 25ng of DNA in a volume of 2µl and set
the tube on ice for ten minutes. (We will provide you with the stock concentration in the lab, you will need to
calculate how much of the stock you will need to proceed – see Pre-lab Question 2 for practice). To the second
tube, add 2 µl sterile dH2O and set the tube on ice for ten minutes. Very gently finger vortex (flick the bottom of
the tube) for a few seconds.

6. Heat shock: Transfer both tubes to a 42°C water bath, heat shock for exactly 30 seconds.

7. Cooling: Transfer tubes immediately back to ice. Allow the tubes to cool for at least 1-2 minutes.

8. Recovery: Under sterile conditions, add 900 µl of LB media warmed to 37°C (this should be pre-warmed in walk-
in incubator) and gently transfer the culture to a round bottom culture tube (sterile glass or disposable plastic
culture tubes provided). This media should not contain antibiotics at this step. Incubate these test tubes 1 hour at
37°C in a rack on the shaking platform in the walk in 37°C incubator. Make sure everything is well labelled including
your initials and lab room! Get into a habit of labelling everything in the lab.

9. Plating post incubation at 37°C:

Label your LB-agar plates with your name, lab day/section and the date. You may wish to even label with your lab
notebook code. It is best to write on the underside of the plate—the part that holds the agar—because lids can
accidentally be switched. You will need the following plates per group:
• 2 LB-kanamycin
• 2 LB

10. You should have two different cultures of chemically competent cells:

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• sample 1 - water control and sample 2- LifeAct-GFP plasmid transformed
• For each of your transformed cultures, spread 100 µL of undiluted culture onto both LB and LB-kan plates
11. Make sure your plates are well labelled with the date, your name, media type, sample label etc.
12. After the cell suspension has adsorbed (~15 minutes), flip the plates agar side up. This way, any condensation that
forms will not drip onto the agar and disrupt colonies. Incubate at 37°C. Colonies should be visible in 12-16 hours.
Best not to leave them longer than 24 hours. We will remove the plates for you the next day. You will observe the
results in the next lab.

13. CLEAN-UP: You have introduced antibiotic resistance genes to bacteria! Dispose of all culture waste (tips, tubes,
bacteria) in Biohazard Waste. This will be autoclaved to destroy potentially dangerous biologics. Do not dispose
of any bacteria or DNA in regular waste or in the sinks. Wipe down your working surface with disinfectant found
in the spray bottles on your bench and put away all equipment. If you should wish to discard any cell cultures at
a future date, place them in autoclave bags or autoclave containers for sterilization.
14. NEXT LAB SESSION: Observe the colonies and record your results. Normally it is recommended to wrap the plate
edges with parafilm, but this won’t be necessary for us. The cells will remain viable at this temperature for at least
a month.

BACKGROUND P1-02
SELECTION OF TRANSFORMANTS:

Typically, low dilutions of transformed bacterial cells are plated on agar in order to isolate individual transformed cells
that contain a recombinant plasmid. Each isolated cell replicates through binary fission to form a colony, and each cell in
the growing colony is a clone of the original transformed cell. Transformed bacteria that contain the plasmid DNA can be
selected for through various techniques. For example, selection can be accomplished by introducing selectable markers
to the plasmid vector. Common selectable markers are generally genes that confer resistance to antibiotics or allow
auxotrophic host cells to grow on minimal medium. The process of selection (ex: presence of antibiotic) ensures the
bacterial cells in each colony outgrowth also retains a copy of the recombinant plasmid. The actual number of plasmid
copies per cell depends on the copy number of the plasmid—that is, the tendency of a plasmid to be replicated so that
there are relatively many or relatively few per cell. Thus, a bacterial culture containing a plasmid with a high copy number
will yield more plasmid DNA than a similar culture containing a plasmid with a low copy number.

The plasmid we are transforming into bacterial cells is called LifeAct-GFP. This plasmid is commonly used to express in
mammalian cells to visualize the actin cytoskeleton in living cells. In order to have enough plasmid DNA to express in
mammalian cells, Plasmid DNA must be replicated in high copies in bacterial cells and purified. The lifeAct-GFP plasmid is
a high copy plasmid that includes the kanamycin resistance gene to confer bacterial cell survival in the presence of
kanamycin (take a look at the plasmid map on the next page). Here are additional references for the use of this expression
vector for visualizing the actin cytoskeleton:

• https://www.ncbi.nlm.nih.gov/pubmed/26317264
• https://www.ncbi.nlm.nih.gov/pubmed/26317264

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It is an important skill to look at plasmid maps and identify the salient features important to its construction. An expression
plasmid (often called an expression vector) such as this, is designed to express a gene product in a host cell, and has
features that any vector may have, such as an origin of replication, a selectable marker, and a suitable site for the insertion
of a gene such as the multiple cloning site. An expression vector must also have the elements necessary for proper gene
expression in host cells. These include a promoter (some plasmids include an enhancer region), the correct translation
initiation sequence such as a ribosomal binding site and start codon, a termination codon, and a transcription termination
sequence. There are differences in the machinery for protein synthesis between prokaryotes and eukaryotes, therefore
the expression vectors must have the elements for expression that is appropriate for the chosen host. For example,
prokaryotes expression vectors would have a Shine-Dalgarno sequence at its translation initiation site for the binding of
ribosomes, while eukaryotes expression vectors would contain the Kozak consensus sequence.

The LifeAct-GFP vector is shown below, and can also be found from the supplier’s website
https://www.addgene.org/58470/

Can you identify some of the important features of this particular expression vector?

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PLASMID PREPARATIONS USING ALKALINE-SDS LYSIS:

The process of preparing plasmid DNA begins with the inoculation of bacterial media with cells from a single bacterial
colony. The initial culture of cells is about 2-10 mL in size. If large amounts of plasmid DNA are required, the initial culture
is used to inoculate a larger flask of media. Table 1 lists the amount of culture required to produce various yields of
plasmid. For our lab, instead of waiting for your isolated colonies to grow, we will have inoculated cultures ready for you
to be able to perform the plasmid isolation. These cultures of Top 10 E.coli cells were transformed with LifeAct-GFP (just
as we will do), and isolated colonies were picked and grown in LB with the selection antibiotic, kanamycin.

Table 1: Plasmid yield vs. culture volume.

Scale of plasmid Yield of plasmid DNA

Culture volume
preparation

miniprep 1-5 mL 100 ng – 5 µg

midiprep 10 mL 20-50 µg

maxiprep 500 mL 1-3 mg

Cells need to be grown to high concentration, and should ideally be in the log phase—the phase of rapid growth. This
ensures high numbers of healthy cells, each carrying plasmid. In the lag phase the culture is no longer dividing as rapidly,
and by the stationary phase, nutrients may be used up, and a larger proportion of cells would be dead. Plasmid yields will
be lower if the cultures are filled with unhealthy or dying cells.

The next step is to concentrate cells from the culture—a centrifuge is used to collect cells into a pellet, and the cell pellet
is resuspended in a smaller volume of buffered solution containing glucose and EDTA. The EDTA chelates divalent cations
like Ca2+ and Mg2+ and serves to destabilizes the bacterial cell wall and plasma membrane. These cations are also required
for nucleases, so the presence of EDTA inhibits enzymatic digestion of nucleic acids once the DNA is exposed. After
resuspension, the bacterial cells are then lysed (broken open) by a harsh chemical treatment with a strong basic detergent
solution (the alkaline-SDS solution). This solution is also strong enough to denature DNA. The alkaline conditions of the
solution are then neutralized by a sodium or potassium acetate solution. This causes proteins to clump together and
precipitate out of solution. The neutral pH also allows genomic DNA to renature in a large insoluble tangle. In contrast,
the two denatured/separated strands of plasmid DNA are able to anneal together again when the solution is neutralized,
but the plasmid DNA remains soluble in the solution. The precipitate, containing protein and genomic DNA, is removed by
centrifugation, and the plasmid containing supernatant is transferred to a new tube. Smaller RNA molecules can also be
isolated in the supernatant using this procedure, so the enzyme RNase is typically added to plasmid preparations so that
RNA molecules may be broken down. RNase may be added to one of the isolation reagents, such as the resuspension
buffer, the alkaline/SDS lysis buffer, or added to the final purified plasmid preparation.

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Next, the Plasmid DNA is precipitated out of the supernatant. Traditionally the DNA would be precipitated out of solution
via ethanol or isopropanol, which disrupts charge screening by water and allows positive ions in the solution to interact
with DNA phosphate groups. This occurs because water forms more non-covalent interactions with alcohol than it does
with nucleic acids, so DNA becomes less soluble in the water/alcohol mixture and precipitates out of solution. Although
low in cost, alcohol-based DNA precipitation can take a fair amount of time even for small-scale plasmid preparations.
More recently, it is becoming standard practice to use commercial plasmid preparation kits to increase the efficiency and
purity of plasmid preparations. Most plasmid DNA isolation kits rely on silica spin columns to isolate DNA. These columns
are thought to depend on the ability of “chaotropic salts” to dehydrate DNA, allowing the DNA to bind reversibly to the
silica via its phosphate groups. A chaotropic agent is a molecule in an aqueous solution that can disrupt the hydrogen
bonding network between water molecules. This effects the native state stability of other molecules in the solution, by
weakening the hydrophobic effect. In this case, the chaotropic salts disrupt the hydrogen bonding between the DNA and
surrounding water molecules, and as a result, interact with the silica column instead. The silica adsorbs the DNA, and
subsequent washing steps allows impurities to be washed away. Water or TE (Tris/EDTA) buffer can be used to elute DNA
off the column.

The Bio Basic Plasmid Miniprep System is one example of a rapid procedure for the isolation of plasmid DNA. Following
cell lysis and neutralization, the cell extract (containing the plasmid DNA) is added to a silica membrane containing a resin
that selectively binds plasmid DNA. The plasmid DNA can be eluted from the resin with an elution buffer. This method
does not require any organic extractions or alcohol precipitations. We will be using this kit in the lab. For more information
about this kit, the product information booklet is posted on Canvas. You will need to refer to this product guide to look
up the volumes of each reagent needed at each step (fill in the blanks in the procedures section).

MEASURING DNA CONCENTRATION

One of the more commonly used practices to quantitate DNA or RNA is the use of spectrophotometric analysis using
a spectrophotometer. Spectrophotometers are used to measure the absorbance of light by a solute. It shines a light of
chosen wavelength through a solution, measures the amount of light that passes through, and reports the amount of light
that has been absorbed by the solution. Quantifying nucleic acids using spectrophotometric analysis is based on the
principle that nucleic acids absorb ultraviolet light in a specific pattern. In the case of DNA and RNA, a sample is exposed
to ultraviolet light at a wavelength of 260 nanometres (nm) and a photo-detector measures the light that passes through
the sample. Some of the ultraviolet light will pass through and some will be absorbed by the DNA / RNA. The more light
absorbed by the sample, the higher the nucleic acid concentration in the sample. The resulting effect is that less light will
strike the photodetector and this will produce a higher optical density (OD). This is also referred to as the absorbance.
Using the Beer-Lambert Law it is possible to relate the amount of light absorbed to the concentration of the absorbing
molecule. In essence, the Beer Lambert Law makes it possible to relate the amount of light absorbed to the concentration
of the absorbing molecule. The following absorbance units to nucleic acid concentration conversion factors are used to
convert OD to concentration of unknown nucleic acid samples:

• A260 dsDNA = 50 µg/ml

• A260 ssDNA = 33 µg/ml
• A260 ssRNA = 40 µg/ml

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Example of Calculation:

A sample of dsDNA was diluted 50X. The diluted sample gave a reading of 0.65 on a spectrophotometer at A260. To
determine the concentration of DNA in the original sample, perform the following calculation:

• dsDNA concentration = 50 μg/mL × A260 × dilution factor

• dsDNA concentration = 50 μg/mL × 0.65 × 50
• dsDNA concentration = 1.63 mg/mL (note this is the same as 1.63 µg/µl)

We will be using microvolume spectrophotometer to measure the concentration of our plasmid DNA. Take a look at the
information from the manufacturer about the NanoDrop 2000. The handbook on measuring nucleic acid concentrations
using the ThermoFisher NanoDrop is also posted on Canvas. https://www.thermofisher.com/order/catalog/product/ND-
2000

PROTOCOL FOR P1-02:

Make sure to write a descriptive purpose for each experiment. An example for this experiment is:
• To isolate, purify and quantify LifeAct-GFP recombinant plasmid from transformed competent TOP10 E. coli cells.

MATERIALS:
2 ml LB media
Stock of Kanamycin
1 LB+Kan plate with colonies of Top 10 E. coli transformants containing LifeAct-GFP plasmid

Liquid cultures of Top10 E-coli transformed with LifeAct-GFP plasmid in LB+Kan

Bio Basic Plasmid Miniprep System
• Resuspension buffer (Solution I)
• Lysis Buffer (Solution II)
• Precipitation buffer (Solution III)
• Spin-columns
• Wash solution (diluted with ethanol prior to use)
• Elution Buffer (2.0 mM Tris-HCl pH 8.0~8.5 or sterile dH2O)
Mini prep silica column
Microfuge tubes for collection of plasmid DNA
NanoDrop2000 Spectrophotometer

PROCEDURE:
Part 1 – Picking colonies:
In P1-01 we will transform E. coli with the recombinant plasmid, LifeAct-GFP in experiment. Instead of waiting for your
colonies to grow, we will have pre-made plates with transformed colonies (DO NOT LABEL THESE PLATES WITH YOUR
NAME). This way you can practice your technique picking single isolated colonies of transformed bacteria and inoculate
LB media containing kanamycin. From there, these inoculated cultures will be grown overnight and stored so you can
observe them next week.

1. Prepare 2mls of LB with kanamycin at a final concentration of (30 µg/ml). Transfer 1ml of LB+Kan into a second
sterile, glass, round bottom culture tube.

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2. Pick 2 isolated colonies, one colony per tube containing 1ml LB+Kan. Incubate in the shaker in the walk-in
incubator at 37C overnight.

Part 2 – Plasmid isolation – Mini prep

1. Each of you will perform one mini-prep.

2. To harvest your cells, add 1.2 ml of each overnight culture to a labeled microfuge tube. Centrifuge the samples for 3
minutes at 8,000 rpm.
3. Remove the supernatants and discard them into the autoclave bags on your bench for tip waste. Add an additional
1.2 ml of overnight culture to the appropriate microfuge tubes. Centrifuge as in step 1 then remove and discard this
supernatant. (You are pelleting more cells on top of the first pellet to accumulate all cells from your culture).
4. You may repeat this to pellet all of your cells (or at least 2-3mls) from the provided culture.

For the appropriate volumes, refer to the instructions for the ThermoFisher PureLink Plasmid Miniprep System.

5. Resuspend the cell pellet in ___100ul_______ of Resuspension Buffer (Solution I) (RNAse A is already added) by
pipetting up and down with a P200 pipette to create a smooth cell suspension. Use a new tip for each pellet. Vortex
briefly to ensure the cells have been thoroughly resuspended.
6. Lyse the cells by adding ___200 ul_______ Lysis Buffer (Solution II) and mix by inverting the tube 4–6 times. The cell
suspension should become slightly viscous. Do not vortex to avoid shearing of chromosomal DNA. Incubate at room
temperature for 5 minutes.
7. Precipitate the DNA by adding ___350 ul_______ Precipitation Buffer (Solution III) and incubate at room
temperature for 1 minute. MIx immediately by inverting the tube 4–6 times. A fluffy white precipitate should form
and the bacterial lysate will appear less viscous. Do not vortex.
8. Centrifuge for 5 minutes at 12,000 rpm to pellet cell debris and chromosomal DNA.
9. To bind the plasmid DNA to the resin in the spin column, transfer the cleared supernatant into the spin column.
Avoid disturbing or transferring the white precipitate.
10. Place the spin column/wash tube into the microfuge and centrifuge for 2 minute at 10,000 rpm.
11. Discard the flow through and place the column back on top of the wash tube.
12. Wash the column by adding 700 ul of Wash Buffer to the spin column and centrifuge the spin column for 2 minute at
10,000 rpm. Remove the spin column from the wash tube, discard the flow through and place the spin column back
into the wash tube.
13. Repeat the wash step.
14. After you discard the flow through, centrifuge the column for an additional 1 minute at 10,000 rpm to remove any
residual Wash Buffer. Discard the wash tube.
15. Place the spin column inside a clean labeled microfuge tube. Elute the plasmid DNA by adding 40 µl of elution buffer
(can use TE or sterile dH2O) to the center of the membrane in the spin column, and incubate for 2 minute at room
temperature. Centrifuge the spin column for 2 minutes at 10,000 rpm. Do not close the cap of the microfuge tube
over the spin column. Point the open microfuge cap towards the middle of the centrifuge. Important! In this step
we will keep the flow through as it contains the plasmid DNA.
16. Discard the spin column. The microfuge tube contains your plasmid DNA.

Determine the concentration of the plasmid using the NanoDrop2000 spectrophotometer.

The DNA yield is determined by measuring the absorbance at 260 nm. The ratio of the readings at 260 nm and 280 nm
(A260/A280) provides an estimate of the purity of DNA with respect to UV absorbing contaminants such as protein. Refer

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to the Thermo Scientific bulletin posted on Canvas for a full description of how the A260, A280 and A230 can be used to
determine the purity of nucleic acids.

1. With guidance from a TA, blank the NanoDrop 2000 spectrophotometer using 2.0 µl of dH2O.
2. Take a reading of your plasmid using 2.0 µl of your plasmid DNA. Record the results in your notebook. Calculate the
concentration of plasmid DNA in your samples. What is the A260/280? What is the A260/230? What are your
conclusions, with respect to the purity of your plasmid DNA?

PRE-LAB QUESTIONS:
1. When you resuspend your bacterial culture in CaCl2, what ratio is the volume of CaCl2 added relative to the original
culture volume?
2. During the lab you will need to calculate how you plan to dilute the plasmid DNA for the bacterial transformation.
Let’s practice: Imagine you are provided with a stock concentration of 100ng/ul. You will use sterile water to
prepare the dilution (and you have as much water/diluent as you need). Come up with a strategy to prepare this
dilution while keeping in mind the calibration limits of your pipetting instruments.

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