Team:Technion Israel/Tar improvements
Introduction
As Tar is the template chemoreceptor of our project, we first need to characterize it in terms of response, movement and location in vivo . In order to do so, plasmid expressing Tar was cloned to chemoreceptors free E.coli strain UU1250. A proper characterization of the bacteria will serve as good comparison for our newly designed chemoreceptors, allowing us to examine if they have the right properties indicating functional chemoreceptors.
Expression
Studies have shown that expression of a sole chemoreceptor in high level increases the sensitivity of the bacteria to the chemoreceptors ligands (1). Due to this property we constructed a high expression
system of Tar chemoreceptor based on K777000 BioBrick. The expression system includes the strongest Anderson promoter (J23100)
and the strongest RBS (B0034),
according to Warsaw 2010's measurement, Tar encoding sequence (K777000) and a double terminator (B0015).
This plasmid, K1992004,
then transformd to UU1250 strain for high expression of single chemoreceptor (fig. 1).
Fig. 1: K1992004 - High expression biological circuit ; J23100 promoter, B0034 RBS, K777000 Tar chemoreceptor and terminator.
In order to optimize the sensitivity of our system we also decided to examine the effect Tar native RBS has on the expression level. 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 expression levels of the two RBS's.
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. Although little is known about the mechanism of
localization, it is important to preserve this property with our designed receptors in order to keep a functional
and 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 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 functionality 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 expressed using B0034 RBS. (a) The cells under white light (b) the cells under 488nm wavelength.
Fig. 2: Tar-GFP expressed using the native RBS. (a) The cells under white light (b) the cells under 488nm wavelength
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 response and movement. In turn these results
will be used as 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 exhibit chemotaxis response and movement 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 suggested higher a sensitivity of the chemotaxis
system, that caused by 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: (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 bacteria express Tar moving toward high concentration of aspartate. As can be seen (fig. 4) after 15 minutes, the number of the bacteria in the frame rose that compared to the control (fig. 5) that remained approximately the same.
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 bacteria express Tar moving away from the high concentration of Co+2. As can be seen (fig. 6) after 15 minutes, the colored bacteria formed a cluster visible to the naked eye, that compared to the control (fig. 7) which did not form cluster. missimg results
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.
