(mellow electric guitar)
Hi. My name is Susan Cohen, and today, we are here
in the Golden Lab at U.C. San Diego.
This video is going to introduce you
to the use of Luciferase reporters in cyanobacteria,
in order to monitor their circadian rhythms.
Cyanobacteria are a phylum of bacteria
that obtain their energy via photosynthesis.
Cyanobacteria are also special,
in that they display circadian rhythms of gene expression,
where the rhythmic expression of up to 80% of their genomes is observed.
A better understanding of the circadian mechanisms
of these simple bacteria could lend great knowledge
and insight into the evolutionary basis
of circadian rhythms, and the means by which
cells can tell time.
In order to visualize the cycling
of rhythmically-expressed genes,
we will first insert a reporter into the genome.
In this case, we are going to use Luciferase.
The species of cyanobacteria we are using today
is synechococcus elongatus PCC 7942,
which can be easily transformed,
meaning that the cells readily take up exogenous DNA.
So first, we have to obtain the genes
that code for the Luciferase enzyme,
as well as its substrate, Luciferin,
from another bacterial species.
These genes are cloned into a plasmid
that will allow them to be expressed in S. Elongatus,
and driven by a promoter sequence of interest.
The promoter sequence chosen will dictate
when the genes are expressed.
This will lead to the production
of the Luciferase enzyme that will react
with the Luciferin substrate, to produce light,
which we can then measure.
The plasmid is placed in the liquid media
with the cyanobacteria, which can take up the DNA
and integrate it into their genome.
Along with the Luciferase and the Luciferin genes,
we also include a gene that creates resistance
to a specific antibiotic.
This allows us to culture the bacteria
on media treated with the antibiotic,
and then only cells that successfully integrate the DNA
we have given them will survive,
because the antibiotic will kill any cells
that did not get the antibiotic-resistance cassette.
Once we have the cells that contain
the antibiotic resistance gene,
and therefore, the Luciferase/Luciferin construct,
we will move them from a plate into our experimental setup.
At this point, we may make other genetic modifications,
such as decreasing or increasing expression
of particular genes, to observe how these alterations
affect circadian expression of the Luciferase reporter.
Today, I will show you four different samples.
First, an unaltered, or wild type sample,
with its clock genes intact.
Second, an arrhythmic mutant.
Third, a long-period mutant,
and fourth, a short-period mutant.
Note that all of these samples came
from the same S. elongatus species,
that had the Luciferase/Luciferin genes transformed.
From this strain, other modifications to the clock genes
were introduced, giving us the different mutants
that we will observe today.
When the reporter gene is expressed,
we will be able to visualize expression
by observing the amount of light produced by the cells.
But first, we need to entrain the bacteria
to a light-dark cycle, so that all of the bacteria
have their clocks set to the same time.
In their natural habitat, the bacteria would entrain
to the environmental cycle driven
by the rising and setting of the sun.
In the lab, we use incubators with timed lights,
to simulate the environment.
This also gives us the flexibility
to plan our experiments without having
to come in at dawn.
Once the bacteria of interest are inoculated
onto a plate where they will grow and be measured
for the duration of the experiment,
they are placed in the incubator,
and left to entrain to a light-dark cycle,
with lights on for 12 hours, and lights off for 12 hours,
otherwise known as LD 12:12.
- Hi, I'm Ryan Shultzaberger, a post-doc at U.C. San Diego,
and I'm gonna walk you through how to gather the data
from the cyanobacterial samples that Susan prepared.
The first thing we need to do is take our entrained bacteria
to an imaging station,
where we can visualize the Luciferase.
Notice that our camera setup is
in a 30-degree Celsius warm room.
This is to ensure that the temperature is constant
throughout our experiment, and that the growth
of the bacteria will not be hindered.
Agar plates containing our samples will be placed
on the turntable, where cutouts have been made
to specifically hold the plates.
Nine plates can be accommodated during each experiment.
Overhead, you will notice a light source
that will keep our samples in constant lighting conditions
for the duration of the experiment.
This is important because cyanobacteria require light
to continue growing.
Once very two hours, the table will rotate each plate
under a CCD camera, to be imaged.
You can see that the plate
that is being imaged by the camera is covered,
and is kept in the dark,
so the weak amount of light produced by the bacteria
can be detected by the camera.
This is important, because otherwise,
the camera will measure light
from the external light source,
which we have placed over the bacteria,
and the bio-luminescent signal will be lost
to ambient light noise.
The CCD camera will detect the amount of photons (i.e., light)
produced over a three-minute period of time.
After imaging, the turntable will rotate
to release the plate back into the light.
Note that we determined experimentally
that these short periods of darkness
every two hours do not affect the clock.
The cells can reset their clocks,
if presented with hours of darkness,
and the rhythms can get messy
if we give a short dark-pulse too frequently.
Both the data and the video are collected
by the computer.
We can then use the data we have acquired
to visualize and analyze the activity
of the Luciferase and the bacteria,
using specialized software.
- Now you will learn how to take raw data
from an experiment, and turn it into a graph
that displays the Luciferase activity
from the cells over multiple days.
Let's take a look at the video we collected
from seven days' worth of data,
taking note that the video has been sped up
for convenient viewing.
We can see that our wild type cells,
highlighted in blue, have an approximately 24-hour rhythm
of gene expression, that we are monitoring
by measuring bio-luminescence.
Highlighted in red, we can see a strain
lacking the core oscillator
that has no rhythm, or is arrhythmic.
In orange, you can see a long-period mutant,
or a strain with a genetic alteration
that causes the clock to run longer than 24 hours.
In green, we can see a short-period mutant,
or a strain that harbors a mutation
that causes the clock to run faster than 24 hours.
Now that we have acquired images of our cyanobacteria,
we can obtain quantitative data
that measures the rhythmicity of expression
of the reporter gene.
The CCD camera we are using is able
to detect the light emission,
by measuring the average pixel intensity
of each strain over time.
This allows the program to clearly show
expression of genes over a selected time period,
as you can see on this graph.
As the colony gets brighter,
the amount of light detected increases.
Then, as the colony becomes dim,
the amount of light detected decreases.
This pattern repeats each day,
as the bacteria follow their cycle.
Let's take a look at the graph in more detail,
starting with the wild type data.
On the Y-axis, we have the amount
of bio-luminescence being produced by the bacteria
measured as pixel intensity.
On the X-axis, we have time in constant light,
also known as LL,
after the bacteria have been entrained
to two cycles of light-dark, or LD 12:12;
12 hours in the light, and 12 hours in the dark.
Here we can see the blue line corresponds
to the wild type data, red to the arrhythmic mutant,
green to the short-period mutant,
and orange to the long-period mutant.
We can see deviations in rhythmicity,
if we observe the peaks in the wild-type strain
and compare them to that of the mutant.
The short-period mutant, which runs slightly faster,
peaks earlier, or to the left of the wild type.
Conversely, if we look at the long-period mutant,
we can see that it runs slightly longer
and peaks to the right of the wild type.
We hope that this laboratory demonstration
was useful to you, and that you now have a better idea
of how circadian biologists go about obtaining
and interpreting data of circadian rhythms in cyanobacteria.
(mellow electronic guitar)