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 chemoreceptorless E.coli strain UU1250. A proper characterization of the bacteria will serve as a good reference for indicating that our newly designed chemoreceptors are functional.
Fig. 1: Scheme of Tar chemoreceptor.
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 high expression of single chemoreceptor, due to the strong promoter and strong RBS (Fig. 2).
Fig. 2: 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 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. 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 swarming assay, in which the chemotaxis ability is examined in a swarm plate. The bacteria depleting the attractant and move outwards, creating a halo. You can view the results in the movement section (Fig. 3).
The results were quite surprising. The strain with native RBS had a better chemotaxis ability, than the strain that contain strong RBS. This finding suggests a higher expression of Tar, means the native RBS might be "stronger" than the strong RBS, according to Warsaw 2010's measurement.
Fig. 3: 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 preserve this property with our designed receptors in order to ensure a functional
and a sensitive chemotaxis response (2).
GFP labeling is a very common way to examine the migration and localization of certain proteins in vivo .
Fusion of GFP (E0040) to Tar
chemoreceptor enabled us to track the migration and localization of the protein to the cell poles as
expected. The fusion was conducted using a flexible linker (J18921)
in order to keep the domain structures of the proteins. The Tar-GFP (K1992003)
expressed using the two expression systems (K1992008 and
K1992009) and examined using fluorescence microscope
(Fig. 1 and Fig. 2). In both cases, high concentration of fluorescence can be seen 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.
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 conducted
to both RBS and native RBS (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 RBS
and the native RBS (Fig. 3). The larger radius of the native RBS suggests a higher sensitivity of the chemotaxis
system, due to a higher expression of Tar (1).
Fig. 1: (a) Tar expression 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. 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 Tar expression using the strong RBS and native RBS in UU1250 strain: (a) Tar expression in UU1250 strain cloned with K1992004 expretion system - strong RBS. (b) Tar expression in UU1250 strain cloned with K1992004 expretion system - Tar native RBS
Attractant response of the Tar receptor tested using chip microscope assay. 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 compared to the control (Fig. 5) bacteria which remained approximately unchanged.
a.
b.
Fig. 4: (a) cells expressing Tar w aspartate t=0 (b) cells expressing Tar w aspartate t=15 min
a.
b.
Fig. 5: (a) cells expressing Tar w motility buffer t=0 (b) cells expressing Tar w motility buffer t=15 min
Repellent response of Tar receptor tested using chip color assay. 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.
a.
b.
Fig. 6: Chemotaxis test on the Chip for Tar UU1250 strain expressing Tar chemoreceptor and Chromoprotein: (a) Repellent added (b) Control- motility buffer.
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.
