Team:Technion Israel/Tar improvements
Introduction
We chose Tar (Taxis Aspartate Receptor) of E. coli as the template chemoreceptor to engineer the chimeric chemoreceptor. To do that, we first need to characterize Tar in terms of location in vivo and its function, mainly the response and movement of bacteria. In order to do so, the plasmid expressing Tar was cloned to an E.coli strain lacking chemoreceptors, UU1250. A proper characterization of the bacteria will serve as a good reference for indicating that our newly designed chemoreceptors are functional.
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Fig.1: a. Scheme of Tar chemoreceptor. b. K1992004 - High expression biological circuit ; J23100 promoter, B0034 RBS, K777000 Tar chemoreceptor and terminator.
Expression
Studies have shown that overexpression of a single chemoreceptor increases the sensitivity of the bacteria to the chemoreceptor's ligands (1). Due to this property we constructed a high expression
system of Tar chemoreceptor, based on the K777000 BioBrick. This expression system includes the strongest Anderson promoter (J23100),
the strongest RBS (B0034),
according to Warsaw 2010's measurement, the Tar encoding sequence (K777000) and a double terminator (B0015).
This plasmid, K1992004,
was then transformed into UU1250 strain to generate UST strain. This new strain is assumed to have a high expression of the single chemoreceptor, due to the strong promoter and strong RBS (Fig. 1).
Fig. 1: K1992004 - High expression biological circuit ; J23100 promoter, B0034 RBS, K777000 Tar chemoreceptor and terminator.
In order to explore the Tar chemoreceptor as it is in nature, we also decided to examine the effect of native RBS, as found in E. coli, on the chemotaxis system. The strong RBS (referred as
RBS) was replaced by the native RBS (referred as nRBS)
of Tar as found in the E.coli genome (Fig. 2). The new expression
system K1992005 differs
only by the RBS, allowing the comparison between the two RBSs.
The two strains, which differ in the RBS (RBS vs. nRBS) were examined with a swarming assay, in which the chemotaxis ability is tested in a swarm plate. In this assay, the bacteria metabolize the attractant and move outwards, creating a halo. Results can be seen in the movement section, Fig. 3.
The results were quite surprising. The strain containing a native RBS had a better chemotaxis ability than the strain containing a strong RBS. This finding suggests a higher expression of Tar, meaning the native RBS is more effective than the strong RBS, according to Warsaw 2010's measurement.
Fig. 2: K1992005 - High expression circuit using the Tar native RBS.
Location
E.coli native chemoreceptors cluster in the cell poles. This property is critical for signal
amplification and adaptation of the cell, since it crucial for additional proteins, such as kinases and adaptors to interact with the chemoreceptor, once it migrated to its proper location in the membrane (for profound information on chemotaxis system, click here. Although little is known about the mechanism of
localization, it is important to retain this property with our newly designed receptors - to ensure a functional
and a sensitive chemotaxis response (2).
GFP labeling is a very common technique to examine the migration and localization of proteins in vivo .
Fusion of GFP (E0040) to the Tar
chemoreceptor enabled us to track the migration and localization of the Tar-GFP fusion protein to the cell poles. The fusion was conducted using a flexible linker (J18921),
in order to preserve the three-dimensional structures of the receptor-proteins. The Tar-GFP (K1992003)
was expressed using the two expression systems (K1992008 and
K1992009), and localization was examined using an inverted fluorescence microscope (Nikon Eclipse Ti, magnification X100, 490nm wavelength)
(Fig. 1 and Fig. 2). In both cases, high concentration of fluorescence can be detected in the cell poles, indicating a proper
migration and localization of the Tar receptor. Comparison between the two expression systems (strong RBS and native RBS)
did not show any significant difference. This finding is consistent with the movement experiments results, which indicate that both strains (with RBS & nRBS) contain an active Tar.
Fig. 1: Tar-GFP fusion results: A. Positive control - E.Coli strain expressing GFP protein. B. Negative control - UU1250 strain expressing Tar chemoreceptor. C. Tar-GFP expressed using B0034 RBS. D. Tar-GFP expressed using the native RBS (488nm wavelength).
Fig. 2: Alignment of Tar-GFP white light and 488nm wavelength results: A. Positive control- E.Coli strain expressing GFP protein, B. Negative control- UU1250 strain expressing Tar chemoreceptor, C. Tar-GFP expressed using B0034 RBS, D. Tar-GFP expressed using the native RBS.
Response and movement
Tar exhibits attraction response toward aspartate and a repellent response away from
Ni+2 and Co+2 concentrations (3). Various chemotaxis assays were performed,
using those substances, to show the bacteria's response and movement. In turn, these results
were used as a reference, in order to test the bacterial behavior with our designed chemoreceptors.
Swarming assay was conducted
with strains that express the Tar chemoreceptor with both RBS and native RBS, respectively (Fig. 1 and Fig. 2). Both exhibited chemotactic response and motility, compared to the
negative and positive control. Moreover, these results show a difference in radius size between the strong RBS, according to Warsaw 2010's measurement,
and the native RBS (Fig. 8). The larger radius of the native RBS suggests a higher expression of Tar, that might raise the sensitivity of the chemotaxis system (1).
Fig. 1: a. Tar expression using (B0034) RBS in UU1250 strain, resulting a halo indicating a functional chemotaxis response. b. Negative control - UU1250 strain without the Tar expression plasmid. c. Positive control - ΔZ strain expressing all chemoreceptors.
Fig. 2: a. Tar expression using the native RBS in UU1250 strain, resulting a halo indicating a functional chemotaxis response. b. Negative control - UU1250 strain w/o the Tar expression plasmid. c. Positive control - ΔZ strain expressing all chemoreceptors.
Fig. 3: Comparsion between our two new strains UST and UNT: a. UST strain - with the plasmid K1992004. b. UNT strain - with the plasmid K1992005.
Attractant response of the Tar receptor tested using chip microscope assay. In this assay, a microfluidic chip was filled with a suspension of bacteria in motility buffer (does not triggers response), and placed under the microscope. The chip was then filled with an attractant, to track the bacteria movement. The strain expressing Tar, is moving toward high concentration of aspartate. As indicated (Fig. 4), after 15 minutes, the number of the bacteria in the frame increased from 49 at the beginning to 120 after attractant was added – more than twofold, compared to the control - – same strain, which treated with motility buffer (does not triggers bacteria response), remained approximately unchanged (55 bacteria at time zero, and 66 after adding attractant) (Fig. 10).
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Fig. 4: a. Cells expressing Tar treated with aspartate t=0 b. Cells expressing Tar treated with aspartate t=15 min.
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Fig. 5: a. Cells expressing Tar treated with motility buffer t=0. b. Cells expressing Tar treated with motility buffer t=15 min.
Repellent response of Tar receptor tested using chip color assay. In this assay, a suspension of colored bacteria is spread evenly in the chip. Following, an attractant or repellent is added to the chip, and the bacteria movement is being tracked The strain expressing Tar, is moving away from the high concentration of Co+2. As can be seen (Fig. 6 a) after 15 minutes, the colored bacteria formed a cluster, which is visible to the naked eye, whereas the control bacteria (Fig. 6 b) did not form a cluster.
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Fig. 6: Chemotaxis test on the Chip for Tar UU1250 strain expressing Tar chemoreceptor and blue-chromoprotein through time: . The bacteria is spread all over the chip. The red arrow represent the location of substance added: a. Repellent was added to the chip's lower entrance. Through time, the bacteria move away from the repellent, creating a dark cluster few inches from the repellent. b. Control - motility buffer was added to the chip's lower entrance. There is no difference in bacteria location through time.
References:
1. SOURJIK, Victor; BERG, Howard C. Functional interactions between receptors in bacterial chemotaxis. Nature, 2004, 428.6981: 437-441.
2. SHIOMI, Daisuke, et al. Helical distribution of the bacterial chemoreceptor via colocalization with the Sec protein translocation machinery. Molecular microbiology, 2006, 60.4: 894-906.
3. BI, Shuangyu; LAI, Luhua. Bacterial chemoreceptors and chemoeffectors.Cellular and Molecular Life Sciences, 2015, 72.4: 691-708.
