Team:Nanjing-China/qs

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In our project, we considered devising our bacteria into some kind of automatic machinery that would be able to sense a target molecule and then assemble to degrade it. To fulfill all these functions, we were bound to design some gene circuits to guide our bacteria.

First of all, we should have an understanding of how bacteria move^[1]. For E.coli, they have flagellar motors to control their movement (Fig. 3-5-1). The direction of rotation of their flagellar motors is controlled by the protein CheY. When CheY is not phosphorylated, the flagellar motors rotate counterclockwise (CCW) and bacteria can migrate. When CheY is phosphorylated (CheY-P), it can bind to the flagellar motorprotein FliM, causing the cell to tumble and thus nonmotile. Phosphatase CheZ dephosphorylates CheY-P and causes the flagellum to rotate CCW. E. coli lacking the cheZ gene (ΔcheZ) cannot dephosphorylate CheY-P, and thus tumble incessantly, being nonmotile. So we only need to take control of gene cheZ to control the motility of our bacteria.

Fig. 3-5-1 Illustration of how bacteria move. CheZ is the key of controlling the motility of bacteria. For wild type, CheZ can be expressed normally, so the flagellar motor alternates between CCW and CW rotation, thus the cell can run or tumble. On the contrary, if there is no CheZ, CheY will remain phosphorylated, causing the flagellar motor rotates CW, thus the cell tumbles exclusively. (Cite by JACS, Shana Toop et al, 2007.)

We would like our bacteria to seek and destroy atrazine in the soil. To achieve this goal, a classic system can do the job (Fig. 3-5-2A). In this system, bacteria can move around randomly but fix when sensing atrazine as the ribosome switch turns on and protein CI is expressed to repress the gene cheZ. As a result, our bacteria would just assemble around the target molecules after a period of time.

However, we can only wait for the bacteria to hang about until they find their targets. Thus it could take a long time before the bacteria assembly and the system may not be a robust design. So we shall spare no efforts to improve it. For better control over the function of our bacteria, we took the quorum sensing system into consideration. We expect some bacteria which first find the atrazine will recruit other bacteria at a distance through some signal so they move towards the target and then fixed at the source of pollution. Therefore we constructed the following gene circuits to realize it (Fig. 3-5-2B).

Fig. 3-5-2 The gene circuits we built to control cheZ. (A) A classic system using CI to repress cheZ. (B) A complex but high-efficiency system using quorum sensing system. (C) A simplified system like the system in (B).

This circuit is composed of three modules: an atrazine-sensing module, a recruiting module, and a brake module. We adopted the quorum-sensing system in Vibrio fischerias the basis for our design. In this system, a small-molecule acyl-homoserine lactone (AHL) synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR^[2]. Our atrazine-sensing module controls the translation of LuxI through ribosome switch which turns on when combined to atrazine. Meanwhile, the recruiting module is composed of a series of elements. In this system, the LuxR-AHL complex promotes the expression of a repressor TetR, which represses the expression of another repressor CI. The gene cheZ is controlled by promoter Pλ, which is repressed by CI. So for those bacteria which first sense atrazine, they send out AHL signal and let the other bacteria receive it. After that, TetR is expressed to repress CI, leading the transcription of cheZ. These bacteria move towards the sender until they sense atrazine and another repressor CI, which is controlled by ribosome switch, expresses as a brake of the movement^[3].

We also design the third gene circuit to simplify the system (Fig. 3-5-2C). This system also consists of the three modules in Pattern 2 and the atrazine-sensing module, as well as the brake module, is exactly the same composition with those two parts. The only difference is in the recruiting module that we use a double-functional promoter Plux/CI. The atrazine-sensing module works just the same way in Pattern 2. In this system, however, the LuxR-AHL complex promotes cheZ transcription via promoter Plux/CI. At the spot where bacteria sense atrazine, AHL will accumulate. The higher AHL concentration is, the stronger transcription of cheZ which means greater motility is, leading to directional movement towards bacteria which send out the signal. Meanwhile, the brake module also functions via promoter Plux/CI. The repressor CI, controlled by ribosome switch which turns on when combined to atrazine, represses the promoter Plux/CI and results in bacteria fixation at source of pollution.

Experiments and Results
At the beginning, we verified gene cheZ in genetic level. We had 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wildtype, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. We extracted the total RNA of these 3 strains and synthesized the 1st cDNA using RT-PCR. Then we did PCR of gene cheZ with the cDNA as template and we also did PCR of 16 sDNA as control. We got the figure through agarose gel electrophoresis (Fig. 3-5-3A). We can see that CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ.

Fig. 3-5-3 Verification of gene cheZ. (A) In genetic level, CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ. (B) In phenotype level, CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate.

Then we needed to verify of gene cheZ from phenotype. We have 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ， CL-M is wild type, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. Then we transformed a plasmid with gene cheZ into CL-1 and got the fourth strain pcheZ.

We inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h and took the photograph (Fig. 3-5-3B). From the figure, we can see that CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate. This indicates that E.coli is nonmotile without gene cheZ or flagella and inducing the expression of gene cheZ can restore motility in a cheZ knockout strain.

In addition, we testified the classic gene circuit.We mix the suspension of bacteria with pre-heated semisolid culture medium and drop solution of atrazine on the plate. Our bacteria assembled around one of the targets after incubation for a period of time (Fig. 3-5-4). It is highly possible that they assembles around the other target after atrazine in the first area is degraded and they are able to move freely again.

Fig. 3-5-4 Our bacteria could just assemble around the target molecules after a period of time.

Finally, we also verified lux system. We constructed a strain with gene circuit as shown in Fig. 3-5-5A from CL-1. This is a lux system without the ribosome switch. AHL synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR. Then the LuxR-AHL complex promotes the expression of a repressor CI, leading to repression of CheZ. So bacteria will assembly at spots where initial density is high in this system. After we inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h, we took the photograph of a stripe pattern (Fig. 3-5-5B), which was formed under the influence of the lux system^[4].

Fig. 3-5-5 Verification of lux system. (A) the gene circuit we constructed. (B) The phenotype of lux system.

For more information of our assembly system, please refer to Model.

Reference
[1] Topp S and Gallivan JP. Guiding bacteria with small molecules and RNA. Journal of the American Chemical Society, 2007, 129 (21): 6807-6811.
[2] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.
[3] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.
[4] Liu C, et al. Sequential establishment of stripe patterns in an expanding cell population. Science, 2011, 334 (6053): 238-241.