Team:Technion Israel/Experiments

S.tar, by iGEM Technion 2016

S.tar, by iGEM Technion 2016


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 metabolize the nutrient in their immediate radius, a concentration gradient is formed causing the chemotactic ability to be initiated leading the bacteria to swim through the pores of the gel and advance towards the higher concentrations of nutrients. As the bacteria progress 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 whether the newly designed chemoreceptor works.
Although this assay is easy to perform, it is not without drawbacks:
(i) The swarming assay is a qualitative and not quantitative test as to the fact that the results are the appearance of the “chemotactic rings” or the lack of them. Other parameters such as the percentage of motile bacteria cannot be derived from it.
(ii) This assay is only suitable for metabolized nutrients and substances. This is caused due to the need for a concentration gradient to activate the chemotaxis-induced swarming, which is created by the consumption of the nutrient described above. This means that the assay is suitable only for chemo-attractants and not chemo-repellents.

For the full protocol, click here.

Fig. 2: A scheme for the swarming plate assay. In brief: Using a low percentage agar media plate, add a drop of bacteria to the middle of the plate. Incubate at 30 degrees for several hours. A halo or “chemotactic ring” should be formed in the agar plate.

Chemical in plug assay

The chemical in plug assay is somewhat similar to the swarming assay, but adds some additional feature to the study of chemotaxis. With this assay the effect of chemo – repellent or non-metabolized substances on the bacterial chemotaxis system can be Investigated.
To perform this assay, a suspension of bacteria in motility buffer is mixed with soft agar (0.3%) at ratios of 1:1 (v:v), and 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 diffuses into the agar leading to the formation of a concentration gradient. 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. 1: Chemical in plug assay of ∆ZraS bacteria (posoitve control). From the top and clock wise: 1M Acetate, 100mM leucine, 1M benzoate, 10 mM α-methyel-asprtate.

Despite it's advantages, this assay proved to be a laborious and challenging task with countless instances of failures. In the full protocol, tips that help tune and reduce the reasons of failure can be found, e.g. losing a lot of bacteria due to the low speed at centrifuging can be overcome by reducing the height of the liquid.
Based on our experience using this method, we were eager to provide easier, faster and better methods for the iGEM community. Hence, we did not conduct this assay as much as other assays.

For the full protocol, click here.

Chip color assay

This assay was used only with bacteria expressing both a S.Tar chemoreceptor and a chromo-protein meant to make the bacteria visible to the naked eye. The purpose of this assay was to test our microfluidic device which is part of the FlashLab system. The chip was designed to concentrate the bacteria, thus enhancing the color gradient formed in response to chemotactic stimulus. In this assay a suspension of colored bacteria was inserted into the chip, following a repellent solution was inserted and the bacterial response was monitored with the naked eye and recorded over a time span of 30 minutes.

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 (Nikon Eclipse Ti) with the ability to record the data as a movie or as a time lapse.
The Use of an inverted microscope was necessary due to nature of the experiments performed which involved use of the FlashLab chip. The structure of the chip made it impossible to view it under the microscope from above.
During our project, two different assays were performed with the microscope:

(1) 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 (repellent: bacteria, v:v) while making sure the final repellent concentration is not lethal for the bacteria. The bacterial response was then recorded as a movie, taking a frame every 50 milliseconds over a time span of 10 minutes.
This assay provided us with a way to ascertain the chemotaxis ability of our engineered strains in real time.

This assay provided us with a simple way to ascertain the chemotaxis ability of our engineered strains in real time. On the down side, it was impossible to test an attractant response using this assay. This is due to the fact that the drop is applied directly on top of the bacteria, encircling the cell, thus weakening the chemotactic response.

For the full protocol, click here.

Video 1: Example of a chemotactic response towards a repellent (Please note: 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.

(2) 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 (attractant: bacteria, v:v). 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.
From the recorded data it was possible to compare between the bacterial response towards an attractant to the response towards a control substance (motility buffer). This comparison indicated whether a chemotactic response has occurred.

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. 1: Trap and Track chip illustration (3)

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.

S.tar, by iGEM Technion 2016