Team:Technion Israel/Experiments
Assays
An abundant number of chemotaxis assays, such as the swarming plate and the capillary assay,
can be found in the literature and has been used in iGEM (2). Throughout this project, we tested, performed and optimized multiple assays to aid with our system’s proof of concept.
Here we present a comprehensive list of assays that can be used for various purposes and in-depth characterization. We categorized these assays into three different scales: Macro, Micro and Nano, leading to three levels of detection: crude, fine and extra fine.
Notably, the nano-scale assay is a novel, chemotaxis assay on a silicon based chip that, to our knowledge, has not been described before and we are happy to contribute this new assay to the iGEM community.
The main assays were as follows:
1. Macro:
a. Swarming assay.
b. Chemical in plug assay.
c. Chip color assay.
2. Micro:
a. Drop test assay.
b. Chip microscope assay.
3. Nano:
a. Trap and track.
Swarming assay
The Swarming assay is a vivid and simple way to study, demonstrate and confirm
the chemotactic ability of bacteria and was first explained by Adler (1,2)
In this assay, a drop of the tested bacteria is added to the middle of a soft agar plate
(0.5%) containing tryptone – a nutrient source. As the bacteria consumes the nutrient in
their immediate radius, a concentration gradient is formed causing the chemotactic ability
to “kick-in” leading the bacteria to swim through the pores of the gel and advance towards
the higher concentrations of nutrients. As the bacteria advances through the gel, “chemotactic
rings” are formed as demonstrated here (2)
Fig. 1: Chemotactic rings of: wild type (MG1655) (left) and bacteria lacking chemoreceptors (UU1250) (right).
In our project, this assay was the first and easiest test performed to verify
if the newly designed chemoreceptor works.
Although this assay is easy to perform it is not without drawbacks. The main
one is that it is a qualitative and not quantitative assay as to the fact that
the results are the appearance of the “chemotactic rings” or the lack of it
and other parameters such as the percentage of motile bacteria cannot be concluded from it.
Furthermore, this assay is only suitable for metabolized nutrients and components.
This is caused due to the fact that a concentration gradient is needed to activate
the chemotaxis derived swarming, which is created by the consumption of said nutrient
as described above.
This fact also means that the assay is suitable only for chemo-attractants but not chemo-repellents.
For the full protocol, click here.
Chemical in plug assay
The chemical in plug assay is somewhat similar to the Swarming assay, moreover, helps with
overcoming some of the swarm plate drawbacks. With this assay the effect of chemo – repellent
or non-metabolized component on the bacterial chemotaxis system can be tested.
To perform this assay, a suspension of bacteria in motility buffer is mixed with soft agar (0.3%)
at ratios of 1:1, then poured into petri dishes to solidify. Following that step, disk shaped
Whatman paper is soaked with the chemical and placed on top of the solid bacterial-agar.
Following incubation, the chemical should diffuse into the agar leading to a concentration
gradient to be formed. This causes the bacteria embedded into the agar to swarm towards or
away from it in accordance to its effect (attractant/repellent) as can be seen here.
Fig. 2: chemical in plug assay.
This assay proved to be long and challenging with countless times of failures, moreover,
better, faster and easier assays were designed and studied, thus it was not conducted as
much as other assays.
For the full protocol, click here.
Chip color assay
The chip color assay was designed to test the FlashLab system, which is meant to show a clear gradient in color in response to chemotactic stimulus. This assay was used only with bacteria expressing both the S.Tar plasmid and a chromo-protein. The assay is straightforward and is sort of a beta test for a FlashLab protocol. A suspension of colored bacteria is inserted into the chip and allowed to rest for several moments to let the bacteria spread evenly, following which a proven repellent is inserted and the chip is recorded over a time span of 30 minutes to view a gradient in the color of the bacterial suspension.
Video 1: experimental results from the microscope assay with bacteria engineered to detect Histamine.
Microscope Assays
Use of a microscope provided us with a relatively simple way to track bacterial chemotaxis
in real time and with clear results. In order to perform the experiment we used an inverted
microscope with the ability to record the data as a movie or as a time lapse.
During our project two assays were performed under the microscope.
Drop assay
The purpose of this assay is to test the bacterial chemotactic response towards repellents.
In this assay, a suspension of bacteria in motility buffer was placed on a microscope slide
and a drop of repellent was added to it at a ratio of 1:5 while making sure the final concentration
of the repellent does not kill the bacteria. The bacterial response was then recorded as a movie.
Since repellent chemotaxis occurs within seconds, this assay provided us with a way to immediately
test the chemotaxis system of our engineered strains.
For the full protocol, click here.
Video 1: Example of a chemotactic response towards a repellent (The black flash is the repellent addition).It is possible to see the bacteria stop swimming in straight long bursts and start tumbling in their place.
Chip assay
The purpose of this assay is to test the bacterial chemotactic response towards attractants.
In this assay, a microfluidic chip was filled with a suspension of bacteria in motility buffer and
placed under the microscope to ascertain the bacteria’s condition (alive and swimming). The chip was
then filled with an attractant at a ratio of 1:6. The bacterial response was recorded as a time lapse,
saving an image of the same location on the chip every 30 seconds for 20 minutes in total.
From the images it is possible to see a rise in the number of cells at a specific location on the chip,
by comparing the response of the bacteria to motility buffer (not an attractant), we can tell if there
was a chemotactic response.
Trap and track assay
Although there is an abundant number of chemotaxis assays available today, most of them were designed
50 to 60 years ago and almost none provide a real time measurement without the use of fluorescence labeling,
for an example FRET test.
The use of Porous Si (PSi) and oxidized PSi (PSiO2) matrices for biological sensing is on the rise. So far
various analytes such as DNA, proteins and bacteria have been proven to be detectable on such matrices. The
common method to monitor the interaction of said analytes within the porous films is reflective interferometric
Fourier transform spectroscopy (RIFTS), as it allows a real time measurement and output for the user.
Here we present the results of an early experiment for the detection of chemotactic activity on the porous
silicon films initially developed for bacterial detection.
Fig. 4: Trap and Track chip illustration (3)
References:
1. Adler, J. Chemotaxis in bacteria. Science 153:708–716.1966.
2. BERG, Howard C. E. coli in Motion. Springer Science & Business Media, 2008.
3. Massad-Ivanir, N., Mirsky, Y., Nahor, A., Edrei, E., Bonanno-Young, L.M., Dov, N.B., Sa'ar, A. and Segal, E., 2014. Trap and track: designing self-reporting porous Si photonic crystals for rapid bacteria detection. Analyst, 139(16), pp.3885-3894.
