To redesign the Tar chemoreceptor we followed the protocol presented in "Rosetta and the Design of Ligand Binding Sites", (1). The purpose of the protocol is to design a binding site around a selected small molecule ligand. The general steps of the protocol can be seen in the flowchart to the right.


Using this protocol we managed to generate a library of mutated Tar receptors that theoretically bind a substance in a novel way and activate the chemotaxis pathway in response to it. For each design we ran 3-5 iterations of the protocol to assure optimal results.

Fig. 1: Flowchart of the ligand binding domain design protocol.

For the purpose of our work, we automated the different steps of the protocol, including the filtering process, turning it into a single main script file complete with well-documented instructions. This script also enables easy modification of the filtering parameters to suit the specific ligand being used in the design. For more information see our software tool.

Histamine is known to be found in decaying food, especially rotten fish. This was our motivation for redesigning the Tar LBD to bind Histamine, as a proof of concept for this computational design technique. This ligand is a derivative of Histidine, which is also an amino acid as Aspartic acid, the native Tar ligand. This increases the chances of a successful result.
Figure 1 and video 1 demonstrate the library for Histamine ligand we obtained after running several cycles and filtration.

Fig. 1: Alignment of Tar ligand-binding domain (LBD). The alignment presents the 11 variants in the library with the native Tar (wild type).

Analysis of the results shows two main regions of mutations, one around amino acid number 34 in the LBD sequence and the second around the 115^th amino acid. These results led us to design and perform a two-step cloning assay, in each step we insert the mutations with single PCR reaction.

Fig. 2: General Histamine-Tar fragment design. Blue lines represent the mutated regions.

Awareness among people regarding lactose intolerance is rapidly increasing. Thus, we want to offer a detection solution using the Rosetta's computational design applications. Therefore, we redesigned the Tar LBD to bind Lactose.
Figure 3 and figure 4 demonstrate the library we obtained for Lactose after running several cycles and filtration.

Fig. 3: Alignment of Tar ligand-binding domain (LBD). The alignment presents the 7 variants in the library with the native Tar (wild type).

Fig. 4: 3D imaging of the 7 variants in the library with the native Tar (wild type), each color represents a different variant.

As this is a novel design of a Tar ligand-binding domain to bind a sugar molecule, we decided to have a proof of concept with a smaller and suitable molecule to bind, a Lactose component - Glucose. Glucose is well known monosaccharide and is the main compound used in the production of energy in living organisms. For this reason we can find existing chemoreceptors for Glucose (2), however redesigning the Tar LBD to bind Glucose was performed as a preliminary step before redesigning it to bind Lactose.
Figure 5 and video 2 demonstrate the library we obtained for Glucose after running several cycles and filtration.

Fig. 5: Alignment of the ligand binding domain (LBD). The alignment presents the 8 variants in the library with the native Tar (wild type).

Analysis of the results shows one main region of mutations, around the 75^th amino acid the LBD sequence. These results led us to design and perform a single PCR reaction to insert the mutations.

Fig. 6: General Glucose-Tar fragment design. Blue line represent the mutated region.

Rohypnol, also known as Flunitrazepam, is used in some countries to treat insomnia. However, it is better known as a 'date rape drug'.
As one application of our project, we would like to offer a simple drug test based on our system to help men and women identify danger when going out. Redesigning the Tar LBD to bind Rohypnol takes us one step closer to achieving this goal.
Figure 7 and figure 8 demonstrate the library we obtained for Rohypnol after running several cycles and filtration.

Fig. 7: Alignment of the ligand binding domain (LBD). The alignment presents the 4 variants in the library with the native Tar (wild type).

Fig. 8: 3D imaging of the 4 variants in the library with the native Tar (wild type), each color represents a different variant

Targeting bacteria towards antibiotics may seem redundant as the bacteria will simply die, however it can be also used as an effective kill switch - a small dose of antibiotics can kill more bacteria if those are attracted to it. To expand our novel approach we redesigned the Tar LBD to bind Ampicillin antibiotics.
Figure 9 and figure 10 demonstrate the library we obtained for Ampicillin after running several cycles and filtration.

Fig. 9: Alignment of the ligand binding domain (LBD). The alignment presents the 13 variants in the library with the native Tar (wild type).

Fig. 10: 3D imaging of the 13 variants in the library with the native Tar (wild type), each color represents a different variant.

The Rosetta’s design process for Histamine ligand produced 870 results, out of which 11 variants remained after filtering. Induction of point mutations was performed on the native Tar LBD in order to build those 11 variants, and out of them only 6 exhibited the expected sequences in sequencing and were subjected to chemotaxis tests. The tests consisted of placing the cloned bacterial solution in a commercial ibidi chip. The chip was then placed under a microscope and the attractant Histamine was added to start the experiment. The interaction of the bacteria with the attractant was observed over time and one frame was taken every 30 seconds for 20 minutes. The control consisted of the same conditions, with motility buffer being placed instead of the attractant, Histamine. Out of 6 tested variants only one (variant number 9) was discovered to be attracted to Histamine. The results of the chemotaxis test for variant number 9 are presented in figure 2.

Fig. 1: Histamine-Tar filtering process scheme.

Finally, showed in video 1 a working prototype of the FlashLab design - a chip that serves as a detection tool based on the chemotaxis system of E. coli bacteria - by using a commercial ibidi chip filled with a suspension of bacteria expressing the novel chemoreceptor and chromoprotein (K1357008). A solution of Histamine in concentration of 10^-3M (the attractant) was added to the chip and the movement of the bacteria was monitored and recorded.

Video 1: from left to right: (1) Histamine-Tar with Histamine atrractent added. (2) Histamine-Tar with motility buffer added (control).