Difference between revisions of "Team:Technion Israel/Design"
m |
|||
| Line 430: | Line 430: | ||
<tr> | <tr> | ||
| − | <td>Reporter genes <b>( | + | <td>Reporter genes <b>(2)</b></td> |
<td>Fusion of a reporter gene to a biological biobrick which its expression is regulated by a material of interest. </td> | <td>Fusion of a reporter gene to a biological biobrick which its expression is regulated by a material of interest. </td> | ||
<td>Requires gene expression and translation, the material of interest has to penetrate the bacterial membrane. </td> | <td>Requires gene expression and translation, the material of interest has to penetrate the bacterial membrane. </td> | ||
| Line 436: | Line 436: | ||
<tr> | <tr> | ||
| − | <td>Chromogenic assay <b>( | + | <td>Chromogenic assay <b>(3)</b></td> |
<td>A chemical reaction which results in the change of absorbance of a certain molecule, when interacted with the analyte.</td> | <td>A chemical reaction which results in the change of absorbance of a certain molecule, when interacted with the analyte.</td> | ||
<td>Specific for a very limited molecules, expensive</td> | <td>Specific for a very limited molecules, expensive</td> | ||
| Line 442: | Line 442: | ||
<tr> | <tr> | ||
| − | <td>ELISA- Immunofluorescence assay <b>( | + | <td>ELISA- Immunofluorescence assay <b>(4)</b></td> |
<td>A specific interaction between an antibody and its antigen is coupled to an enzymatic fluorescent reaction.</td> | <td>A specific interaction between an antibody and its antigen is coupled to an enzymatic fluorescent reaction.</td> | ||
<td>Specific antibodies are required, expensive, requires special equipment</td> | <td>Specific antibodies are required, expensive, requires special equipment</td> | ||
| Line 448: | Line 448: | ||
<tr> | <tr> | ||
| − | <td>HPLC <b>( | + | <td>HPLC <b>(5)</b></td> |
<td>Chromatographic separation based on polarity.</td> | <td>Chromatographic separation based on polarity.</td> | ||
<td>Requires special equipment requires expertise,expensive</td> | <td>Requires special equipment requires expertise,expensive</td> | ||
| Line 502: | Line 502: | ||
<p class="references"> | <p class="references"> | ||
References:<br> | References:<br> | ||
| − | 1.MAZZAG, B. C.; ZHULIN, I. B.; MOGILNER, Alexander. Model of bacterial band formation in aerotaxis. Biophysical journal, 2003, 85.6: 3558-3574.<br><br> | + | 1. MAZZAG, B. C.; ZHULIN, I. B.; MOGILNER, Alexander. Model of bacterial band formation in aerotaxis. Biophysical journal, 2003, 85.6: 3558-3574.<br><br> |
| − | 2. Santos-Figueroa, Luis E., et al. "Chromogenic and fluorogenic chemosensors and reagents for anions. A comprehensive review of the years 2010–2011." Chemical Society Reviews 42.8 (2013): 3489-3613.<br><br> | + | 2. Naylor, Louise H. "Reporter gene technology: the future looks bright."Biochemical pharmacology 58.5 (1999): 749-757.<br> |
| − | + | 3. Santos-Figueroa, Luis E., et al. "Chromogenic and fluorogenic chemosensors and reagents for anions. A comprehensive review of the years 2010–2011." Chemical Society Reviews 42.8 (2013): 3489-3613.<br><br> | |
| − | + | 4. Blake, Christopher, and Barry J. Gould. "Use of enzymes in immunoassay techniques. A review." Analyst 109.5 (1984): 533-547.<br><br> | |
| + | 5. Kucharska, Marta, and Jan Grabka. "A review of chromatographic methods for determination of synthetic food dyes." Talanta 80.3 (2010): 1045-1051.<br><br> | ||
</p> | </p> | ||
Revision as of 14:05, 19 October 2016
Introduction
FlashLab is a novel detection tool based on the chemotaxis system of E. coli bacteria. It utilizes chemotaxis to concentrate bacteria expressing a chromo-protein, this in turn, creates a visible gradient in color – detection of a target material.
FlashLab is an application of the S.Tar project. S.Tar is a platform for programmable chemotaxis which allows the user to select the material that will induce a bacterial chemotactic response. For more information please visit S.Tar page.
Using S.Tar technology, FlashLab can detect a variety of materials: hormones, amino acids, organic compounds etc.
Fig. 1: A scheme of the FlashLab concept, add bacteria expressing the chemoreceptor of your choice and a chromo protein, to a fluidic chip. Add the sample in question to said chip. If the sample contains the substance that is recognized by the chemoreceptor, a displacement of the bacteria will become visible. If not, then no displacement will be seen.
Fig. 1:The geometry of a commercial fluidic chip.
The chip is open on the bottom part and closed with a standard microscope cover glass (0.3 [mm] thick).
FlashLab setup
The setup of the device is composed of two parts, as shown below (Figure 2):
a. The channel is filled with colored E. coli bacteria suspended in motility buffer.
b. The sample is loaded into one of the entry slots.
Fig. 2: The chip setup.
FlashLab test
Once the sample is loaded, it diffuses into the channel. If the sample contains a repellent, the bacteria will react and flee from it as shown below:
Fig. 3: Chemotaxis reaction in the chip.
The chemotactic response will result in visible changes in the bacterial concentration throughout the chip: Very low concentration in the immediate area of the slot in which the sample was loaded, adjacent to a higher concentration of bacteria created due to the fleeing bacterial population. These changes will be visible to the naked eye, as the higher concentration of colored bacteria results in darker color (blue gradient, figures 2 and 3).
If the sample does not contain the target material, the bacteria will not react and no gradient will be formed.
For more information see mathematical model.
Results
FlashLab parts
The setup for the device is as shown in "Design and Implementation". The bacteria, UU1250 E.coli strain with a cloned Tar-PctA receptor , was picked from a petri dish and suspended in motility buffer. The Chemo-repellent used was TCE in concentration of 0.02 [M] while the control was motility buffer. The pictures were taken in a differential of 5 [min] apart.
Fig. 1: Chemotaxis of E. coli with a S.Tar PctA receptor due to exposure to TCE (enhanced picture).
After 20[min] , a noticeable color gradient is formed in the channel. After 360 min a relatively light shade can be seen near the entrance. adjacent to it, an area with a darker tone and from there up to the end of the channel, the color did not change at all. This fits the theory perfectly, as the lighter shade is caused by colored bacteria moving away from the repellent. The darker shade is the clustering of bacteria in the chemo-repellent diffusion limit. All other bacteria in the channel, were not exposed to the repellent and did not react accordingly.
For more information see chip experiment protocol
Conclusion
We developed FlashLab, the hardware of the S.Tar platform. S.Tar has the potential to offer a wide library of “expert” strains for the detection of materials that do not naturally induce chemotactic movement in the native E. coli. This was obtained using synthetic biology tools, including computational design which allowed us to create new mutations in the Tar chemoreceptor.
FlashLab is a tool which combines both biological and mechanical aspects. As we mentioned before, computational tools were required as well in order to develop the S.Tar platform. This multidisciplinary innovation was achieved thanks to the different backgrounds of our team members. Our team is comprised of students from the faculty of Biotechnology who were in charge of the Tar chemoreceptor modification in the wet lab, students from the faculties of Mechanical engineering and Chemical engineering who developed the mathematical model for the flow and chemotaxis profiles inside the microchannel and students from the faculties of Electrical engineering and Computer Science who were in charge of the computational design aspects of the project. The unique composition of our team was crucial to the construction of a complex but elegant system.
Advantages
There are several methods for detection of small molecules. The following table summarizes the most common methods in the field:
| Method | Description | Disadvantages |
|---|---|---|
| Reporter genes (2) | Fusion of a reporter gene to a biological biobrick which its expression is regulated by a material of interest. | Requires gene expression and translation, the material of interest has to penetrate the bacterial membrane. |
| Chromogenic assay (3) | A chemical reaction which results in the change of absorbance of a certain molecule, when interacted with the analyte. | Specific for a very limited molecules, expensive |
| ELISA- Immunofluorescence assay (4) | A specific interaction between an antibody and its antigen is coupled to an enzymatic fluorescent reaction. | Specific antibodies are required, expensive, requires special equipment |
| HPLC (5) | Chromatographic separation based on polarity. | Requires special equipment requires expertise,expensive |
FlashLab advantages as a detection tool:
- Cost effective The only major cost is the fluidic chip which costs about 15$ and can be reused multiple times.
- Fast as shown in the experiments, the detection takes about 30 minutes. This is faster than most other bacterial detection (based on transcription and translation) and most laboratory tests (HPLC).
- Versatile The S.Tar system enables this device to detect a variety of materials: hormones, amino acids, organic compounds etc.
- Sensitive Bacteria can sense extremely low concentrations of target material.
- User friendly The setup of the system is an easy two part process. To reuse the chip the user required to flush the channel with water and to dry it up.
In comparison, Flashlab, although not the most accurate, offers a fast, cheap, easy to use and versatile detection.
References:
1. MAZZAG, B. C.; ZHULIN, I. B.; MOGILNER, Alexander. Model of bacterial band formation in aerotaxis. Biophysical journal, 2003, 85.6: 3558-3574.
2. Naylor, Louise H. "Reporter gene technology: the future looks bright."Biochemical pharmacology 58.5 (1999): 749-757.
3. Santos-Figueroa, Luis E., et al. "Chromogenic and fluorogenic chemosensors and reagents for anions. A comprehensive review of the years 2010–2011." Chemical Society Reviews 42.8 (2013): 3489-3613.
4. Blake, Christopher, and Barry J. Gould. "Use of enzymes in immunoassay techniques. A review." Analyst 109.5 (1984): 533-547.
5. Kucharska, Marta, and Jan Grabka. "A review of chromatographic methods for determination of synthetic food dyes." Talanta 80.3 (2010): 1045-1051.
