Week 4: Transformation Results and Spectrophotometry
Transformation Results
if you were doing all parts of the experiment yourself, you would pick a white colony from the insert plate to grow in liquid culture and then measure in the spent media
you will be creating a table of your personal colony counts and calculating transformation and ligation efficiency during lab
this is done for you behind the scenes
resulting isolate plasmid will be used for plasmid mapping
Transformation Results OLM
we performed a restriction digest and ligation in lab 1 to get insert into our plasmid
transformed the recombinant plasmid into E.coli JM101 so the bacteria could express our insert
the transformation was plated and incubated overnight at 37 degrees C
plates kept at 4 degrees C until lab
Technical vs. Experimental Controls
technical: controls that are specific to the technique (i.e. restriction digest, ligation)
ensure that the technique is working as it should
getting expected results; no interference giving us something we shouldn’t be getting
experimental: control that represents the “normal” condition (if trying something new for e.g.)
what you compare your experimental sample to
can be considered one of the experimental conditions
if certain concentration makes a technique better/improves disease outcome — different concentrations would be different concentrations & no substance would be control (and also one of the conditions)
not all experiments have an experimental control
which plasmid is better at transforming — no normal plasmid (no experimental control)
all of our plates were either a sample of interest or a technical control
sample of interest: “insert” plate
plate that we’re actually going to work with
will do downstream applications with
not a control — one we’re interested in
we are not comparing this sample to anything else, but if we were, it would be the experimental sample
would be experimental sample if comparing the effectiveness of different transformation protocols for e.g.
white colonies contain the insert (looking for the white colonies)
operon disrupted by insert (contain the insert)
will see a mix of white and blue colonies
ligation technical control: “H2O” plate
water added instead of insert
must keep volume equal
always want to replace substance with neutral one
did not just add “nothing”
expect no ligation of insert into MCS so all blue colonies
showing that the only wat to get white colonies is if there is an insert present
transformation technical control: uncut plasmid added to E.coli
expect transformation to happen into competent E.coli
expect blue colonies — no insert
makes sure that the bacteria are able to be transformed by this particular plasmid
transformation technical control (another): TE buffer tube (not diluted)
TE buffer = liquid that the plasmid is floating in when given to you
by adding just TE, no plasmid was added to the E.coli during transformation
adding TE but NO PLASMID (E.coli don’t get plasmid during transformation
plasmid to be digested was floating in TE buffer
plated on LB + ampicillin plate
expect no colonies — don’t have gene to be able to grow in presence of ampicillin
makes sure we don’t get growth if we aren’t supposed to get growth
not contaminated
not mixing up samples
transformation technical control (and another!): TE buffer (no plasmid) added to E.coli but plated on LB only plates
no ampicillin on plate
expect growth
makes sure that the E.coli are alive and did not get killed during transformation protocol
dilution because we want to be able to see individual colonies
not too many bacteria
if did not dilute, would get a lawn of bacteria = confluent growth
diluted to get countable number of colonies
Controls MUST show Predicted Results
if ANY of your technical controls do not show what is expected, you cannot trust your samples
you cannot analyze data or draw any conclusions
you cannot use sample of interest in downstream applications
What if plasmid does not ligate to insert or to self?
plasmid not ligated = linear
when linear plasmid enters bacteria, exonuclease will degrade linear DNA from end (no longer in plasmid)
In lab:
you will be counting the colonies and creating a table of results
table should include:
sample name (i.e. insert, H2O, TE, etc.)
plate type (LB or LB + amp — include amp concentration 100 ug/mL)
number of white colonies
number of blue colonies
you will then calculate your transformation and ligation efficiency
you will present and discuss this in your lab report
include the transformation and ligation efficiency in Benchling
if transformation did not work, present that in lab report with explanation of what went wrong (no marks lost)
Transformation Efficiency (TE) Calculation
TE = (# white + blue colonies on insert plate) / (total colonies LB plate)
= 12 / 85 × 10 ^6
= 12 / 8.5 × 10^7
= 1.41 × 10^-7
transformation efficiency — no units! not percentage! just ratio!
# white + blue colonies on insert plate = number of bacteria/colonies that were transformed with plasmid
e.g. 5 white & 7 blue
total colonies on LB plate (TE diluted) = total number of bacteria we started with
e.g. 85 white & 0 blue
must take dilution into account
multiply by 10 ^ +6
only concerned with number of colonies for Insert plate and diluted TE plate
Ligation Efficiency Calculation
LE = (# white on insert) / (all colonies - white + blue)
= 5 / 12
= 0.42
ligation efficiency — no units! not percentage! just ratio!
only concerned with insert plate (only plate with insert — only one that underwent ligation)
proportion of plasmids with insert ligated / plasmids that were transformed into bacteria
# white colonies in insert = 5
# blue colonies in insert = 7
Why Do My Colonies Look Funny? Satellite Colonies
might see that the colonies look funny — small dots around colonies
satellite colonies: commonly seen with ampicillin resistance
only large colony is “real”
occur because of the way the ampicillin resistance is created
transformed bacteria with plasmid with amp resistance cassette
satellite colonies are not considered real colonies — do not include in counts!
occur because of the way the ampicillin resistance is created
how does ampicillin work
ampicillin is bactericidal: kills bacteria
inactivates transpeptidases - cross link peptoglycans (in cell wall)
disrupts cell wall synthesis in actively growing cells leading to lysis
any new bacteria that are being formed cannot create a structural cell wall
end up lysing
they die
Ampicillin Resistance Gene: codes for Beta-Lactamase
cleaves a structural ring in ampicillin molecule itself
ampicillin loses its function — cell walls will be able to be formed in newly formed bacteria
results in loss of function of ampicillin
secreted by bacteria
can destroy the ampicillin surrounding the colony
allows non-transformed colonies (satellite) to form
leaves the bacteria — destroys ampicillin surrounding colony of bacteria
non-transformed colonies (satellite) form
white fog: high number of satellite colonies
big juicy white colony in middle surrounded by fog
sometimes there are fewer satellite colonies — could count them but DONT
commonly see satellite colonies in research
Transformation Efficiency
number of colonies on insert plate / number of colonies on DILUTED TE plate
# on insert plate is ANY colour (blue and white)
# on diluted TE plate is colonies on LB plate
need to account for dilution factor (10^-6)
multiple # of colonies by 10^6
record this number in your lab book
even if we overcome barriers, transformation is still a rare event
Ligation Efficiency
use insert plate only
# white colonies / (# blue + white colonies)
record this number in your lab book
use personal numbers (from lab) in lab report
Spectrophotometry
1: Principles of Spectrophotometry
measure the concentration of something in a sample (e.g. concentration of insulin)
absorption spectroscopy: using light to probe matter
how a sample absorbs light
lambda = wavelength (correspond to different colours)
molecule will absorb wavelength strongly (others won’t)
linear?
P sub n = incoming power
high intensity, lots of photons
material will absorb a lot of light
small amount of power out
lower concentration: more outcoming power (absorbs less light)
ratio of output power to input power means something
% transmittance: output light/input light x 100%
spectrophotometer: measures power in and out (specifically for visible light spectrum)
absorbance: A (unitless quantity) = 2 - log(%T)
A = 2 - log(50) = 0.3
for 50% transmittance?? absorbance?
use wavelength that is absorbed by molecules but not others
measure absorbance spectrum (A vs. wavelength)
when absorbance is high, a lot of light is absorbed
peak of graph is the lambda max (short wavelength for this example); means a lot of light is being absorbed by the molecules in the sample
use this for spectroscopy experiment
typically choose this one; most bang for buck
a high concentration of a substance that absorbs a given wavelength will also have a high transmittance
2: Spectronic 20
A) How does a Spectronic 20 work?
spectrophotometer: measures the amount of light that gets transmitted
source → monochromator → sample → detector
source = tungsten filament light bulb — heats up & gives off white light
white light goes through slit (narrow band) and pass through prism or other devices
separate all different colours of light
can rotate prism to sweep different colours of light back and forth over exit slip — select specific wavelength
shines that wavelength through sample
some light get’s absorbed, some gets transmitted
detector converts the amount of light intensity into electrical signal that we can then amplify and measure
nanometers (nm) for visible light
B) How to Operate a Spectronic 20
how to generate an absorption spectra using a dye called DCP
this is a basic spectrophotometer with analog dials and manual setting of 0% transmittance and blanking
Spec 20s accept glass test tubes in the sample chamber
requires a high volume of sample
more advanced spectrophotometers: use same principles
automatically set 0% transmittance
automatically blanks
uses cuvettes (much smaller sample volume)
100 uL — 2 mL
a bit faster
light that emits white light and bounces off diffracting gradient then deflected back at sample
wavelength camera measures wavelength at sample
can pass different energies of light through sample and measure absorbance
make absorption curve
use left on to turn on (clockwise) — 15 minutes on so lamp can warm up
use left knob to bring red meter to 0 percent transmittance
2 scales: top is percent transmittances, bottom is absorbance
top: zero
bottom: measurement
0% — mirror: can’t see the reflection in the mirror = correct level
blank should contain everything except the substance you are trying to measure
we are measuring 2 dyes dissolved in DI (distilled) water so the blank is DI water with no dye
right knob to 100% transmittance (no mirroring of dial)
told the spectrometer what nothing is and what everything is
filter at bottom: gives range
make sure it’s in the proper range (flips back and forth)
wouldn’t be able to zero spectrophotometer
take out DI water blank and create absorption spectrum
put in red dye: will flip to wherever (0.084)
if you can see the meter in the mirror, you are NOT perfectly lined up to read the value
three decimal places
next measurement: 0.053
repeat by changing the wavelength,
must re-blank every time you change the wavelength (must zero and do 100% transmittance)
greatest reading for absorbance
taking measurements at 20 nm intervals (between 360 and 600)
depending on where absorption is highest, is the lambda max
use lambda max for standard curve
3: Microplate Reader
spectronic 20: glass tubes and requires large sample volume of 3 mL — limited to 1 sample at a time
digital spectrophotometer: use glass/disposable plastic cuvettes
sample volume of 1 mL - 100 uL volume, 1 sample at a time
both read in a horizontal manner; light path is horizontal
microplate reader:
put samples on microplate
microplate is loaded into drawer
drawer will close and load the plate into the machine
each well is positioned under/over a lamp one-by-one automatically by the machine to acquire reading
can be read from top or bottom (read vertically)
96 well plates are common → 96 samples!
clear, white, or black
small volume of 100-200uL
Modes of Operation (most microplate readers have 3)
Absorbance: spectrophotometer
microplate reader will work in the same way as a spectrophotometer
for multiple samples at the same time
light from lamp → filter → collect wavelength of light → through each sample → transmittance measured and converted to absorbance reading
Fluorescence:
light from lamp → filter (select wavelength) → hit each well & sites fluorescence tag that is in each sample
fluorescent tag will give off different wavelength of light → filter & select for detection of that particular wavelength
detected by P & T photomultiplier tube
converted into numeric reading
Luminescence:
light that is naturally given off by sample (bioluminescence) passes through filter and is detected by P & T (photomultiplier) tube and converted into numeric reading
Applications: Quantification
create a standard curve just as with a spectrophotometer (serial dilution)
all standards and unknowns (samples) are loaded on the same plate
need to include each standard and sample should be loaded in triplicate
technical replicate
measure of precision
different from biological replicate
use the average of the triplicates to create standard curve and interpolate sample concentration
difference: rather than reading each standard & sample one at a time — all of the standards and unknowns on one plate
when using instrument, must be loaded in triplicates
triplicates = technical replicate (3 different readings of same sample)
measure of precision of instrument
very different from biological replicate: measuring distinct samples
when analyzes, use average of triplicates and determine sample concentration of average from each sample
Applications: Colorimetric Assays
similar to standard curve but is a relative quantification
do not have standards
measure things like: cell viability, cell proliferation, cytotoxicity
example is MTT assay
quantification application can also often be colorimetric
colorimetric = change in colour
4: Standard Curve
overview of the methodology needed to create a standard curve
will be creating the standard curve and measuring the same standards and samples with 2 methods: Spec 20 & Plate Reader
you will be measuring the amount of insulin in the spent media of our transformed E.coli JM101
Does having the INSR insert change the amount of insulin found in the media?
using both the spectrophotometer and plate reader to make the standard curve
standard curves: very commonly used technique to quantify (concentration etc.) an unknown sample
start with sample of unknown concentration + standard with known concentration
start by making a dilution series of your standard — serial dilution method
serial dilution of known standard
transfer the dilution series to a cuvette or test tube and measure the absorbance of each standard
transfer to spec tube (or cuvette) or the microplate
measure absorbance of each standard
take standards and absorbances and graph them
absorbance vs. concentration
create line of best fit — give an equation of line — determine where sample falls in the standard curve
if the sample is outside the range of the standard curve
if unknown is below or above — can’t use standard curve
if in the x range, can interpolate
cannot extrapolate, can only interpolate
R² value will give indication of how accurate line is
R² is 0.93 — not the best but not bad
evaluating whether you can trust
Our Experiment
we are examining the concentration of a substance of interest found in spent media
spent media: media that E.coli have been grown in for 24 hours
can measure amount of substance depleted from media or amount of substance secreted by bacteria into the media
which one you are measuring depends on class experiment (OWL)
transformed E.coli JM101 with plasmid with INSR insert put into it
white colony will be picked (has insert) and grown in liquid culture
liquid = media
colorimetric assay to measure concentration of substance of interest
colorimetric assay: just means there is a colour change
lambda max cannot be directly measured by spectrophotometer
increase of colour means increase in substance of interest
specific assay we’re using (not really the colorimetric)
Labrador retriever is a dog (dog = colorimetric)
details of the assay (name, manufacturer, lambda max)
standard curve was created with the standard provided in the kit
create serial dilution with knowns
have unknown samples here
one reagent in kit is a colour reagent — add it to everything (unknown and standards)
solutions will turn a colour depending on concentration that’s in there
we are indirectly measuring the substance of interest
will be creating a standard curve which will be measured using both the Spec 20 and the plate reader
one set of serial dilutions, one set of unknown samples
use them both in the Spec 20 and plate reader
use the standard curves to calculate the concentration of our substance of interest in the spent media
info will be included in your lab report
USE THE EXCEL SPREADSHEET TO GENERATE AN EQUATION OF THE LINE
check with answer spreadsheet