Team:Uppsala/Project Targeting

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The assembly plan of the Targeting system.

Background

Movement and Chemotaxis

Chemotaxis is the cells system for directed movement which allows the cell to move towards nutrition sources as well as away from toxic substances. The chemotactic system is a complex chain of phosphorylation reactions. At the end of the chain is the flagellum-binding protein CheY. The phosphorylated CheY (CheY-p) binds the flagella and causes the cell to tumble in place, essentially remaining immotile. The phosphatase CheZ dephosphorylates CheY, causing CheY to unbind to the flagella, resulting in directed movement of the cell [1]. More factors are involved in the full chemotactic pathway of E. coli, although they are not of importance for our project.

Using Chemotaxis to track down a pathogen

Due to the complexity of the chemotactic pathway of E. coli, controlling the steering of this chemotactic car was not an option. Instead, we decided to manipulate the breaks, so that our bacteria would stop moving once close enough to the pathogen and deliver a deadly dose of bacteriocin.

To achieve this stop-and-kill mechanism, focus was laid on the protein CheZ. As mentioned above, the bacteria cannot move straight in the absence of CheZ, but tumbles in place instead, so by stopping the production of CheZ we essentially get immotile bacteria. Then, by producing CheZ under controlled manners, the bacteria would ideally become motile again but on our terms. The production of CheZ would be controlled by a system based on the quorum sensing (see the Sensing system) of our pathogen, designed by the Sensing group. The result would be low production of CheZ when close to the pathogen and high production when far away from it.

System design

The ultimate design of our system was the CheZ gene combined with our regulatory Sensing system. However, merging systems is quite a big first step so we decided to divide our work in stages where the final stage was CheZ and the Sensing system combined.


Stage 1

As our goal was to restore motility by inserting CheZ we obviously needed a cheZ-knockout strain of E. coli. We speculated whether we could create such a strain ourselves using various gene-deletion methods but this would require a lot of work on its own. Instead, we received two cheZ-knockout strains and one motile “wildtype” strain as a kind gift by Prof. Parkinson (see Acknowledgements). The two cheZ-knockout strains were called RP1616 and UU2685 and the wildtype RP437. Note that we could not use the standard E. coli lab strain DH5-alpha for our experiments, since it is lacking flagella [3].

The final stage, were the Targeting system is combined with the Sensing system. In the absence of Yersinia enterocolitica, the activator YenR will interact with the yenbox and induce the expression of cheZ..

Stage 2

Since we did not know how different expression levels of cheZ would affect motility, we decided to combine cheZ with constitutive promoters of different strength to see if we could find the optimal expression level of cheZ. The promoters were all from the Anderson family of constitutive promoters available in the iGEM registry of parts. The promoters were the following, with their relative strength within parentheses:

J23100 (2547)

J23113 (21)

J23114 (256)

The same RBS (BBa_B0034) was used for all constructs.

Stage 3

This final stage was simply our regulatory system, the Sensing system, combined with the RBS and cheZ. The idea was that transcription of cheZ would be increased when far away from the pathogen and reduced when close to the pathogen.

Result

All motility tests were carried out on tryptone swarm plates (10g/L bactotryptone, 5g/L NaCl, 2,5g/L agar) based on a protocol from 2011’s WITS CSIR team [2]. Overnight cultures were grown to mid-log phase (OD600 = 0,5) in LB medium and then inoculated on swarm plates.

In figure 1, a selection of the results of our swarm plate assays is presented.

Figure 1. Swarm plate assays. 1A, 1B and 1C were performed on the same batch of swarm agar plates as 1D, 1E and 1F, respectively.

The following strains are present in the figure:
  • 1) B0034_J23100-cheZ (UU2685)
  • 2) B0034_J23113-cheZ (UU2685)
  • 3) negative control (UU2685 with empty pSB1C3 plasmid)
  • 4) positive control (RP437 with empty pSB1C3 plasmid)

In 1A, 1B and 1C, strain no. 1 has been inoculated in the top section. In 1D, 1E and 1F, strain no. 2 has been inoculated in the top section. On all plates, strain no. 3 has been inoculated in the bottom-left as a negative control, while strain no. 4 has been inoculated in the bottom-right as a positive control.


In the swarm plates that are presented in figure 1, only four of the positive controls (strain 4) showed signs of motility. In 1D, strain no. 2 is swarming. This was the only detected instance of swarming for these fifteen swarm plate assays (three replicates of assays for five different strains). Based on the absence of swarming in the positive controls, we concluded that the swarm plate assay was not reliable.
To improve the performance of the swarm plate assay, we changed the culturing conditions to prevent the swarm plates from drying out. Swarm plates were directly inoculated from restreaks or from frozen stocks, wrapped in parafilm and then incubated at room temperature for two days. The results of these extra motility plates are presented in figure 2.


Figure 2. Swarm plate assays performed at room temperature. Strain 1 and 2 were inoculated from restreaks, while strain 3, 4 and 5 were inoculated from frozen stocks.

The following strains are present in the figure:
  • 1) positive control (RP437 with empty pSB1C3 plasmid) [220]
  • 2) negative control (UU2685 with empty pSB1C3 plasmid) [216]
  • 3) B0034_J23100-cheZ (UU2685) [170]
  • 4) B0034_J23113-cheZ (UU2685) [171]
  • 5) B0034_J23114-cheZ (UU2685) [172]

On all plates presented in figure 2, positive controls (strain 1) swarmed, while negative controls (strain 2) did not swarm. The altered protocol for the swarm plate assay was thus able to distinguish motile strains from non-motile strains.

Strain 3, containing a cheZ expression cassette with the J23100 promoter, swarmed all six times when it was tested, though not to the same extent as the positive control. Strain 4, containing a cheZ expression cassette with the J23113 promoter, also showed to be motile, although it only swarmed to a small extent.

The result of the strain containing the cheZ expression cassette including the J23114 promoter (strain 5) is not as clear as that of the other two constructs. The structures that have grown upon incubation resemble the negative control. For strain 5, these structures include cloudy formations, however, which is most clearly visible in figure 2C.

Based on these results, we conclude that both the cheZ expression cassette including a J23100 promoter and the cassette including a J23113 promoter were able to restore motility in a cheZ mutant. It is not clear if an expression cassette containing J23114 was able to achieve the same result. Further experiments should be performed to determine this.

The expression cassette including the Anderson promoter J23100 swarmed to a greater extent than the cassette including the Anderson promoter J23113. This was to be expected, as J23100 is a stronger promoter than J23113. The strain containing the expression cassette including J23100 swarmed to a smaller extent than the positive control, however, which is surprising, given the fact that J23100 is such a strong promoter.

This could be caused by the fact that the number of cells that was applied during inoculation was not normalized by growing appropriate cultures to mid-log phase. The positive control might simply show more swarming, because of a higher cell count. Including the protocol for growth in further tests could show if this is the case.

Wolfe and Howard[4] give another possible explanation. They state that cells that tumble infrequently may move slower on agar plates, as they alter their direction only slowly after running into an agar molecule. A cheZ expression level that is high might in this way restrain swarming, just like a low expression might hinder it. Coupling more Anderson promoters in cheZ expression cassettes, might help find the optimal promoter strength for swarming.

Parts

Fav.BioBrick codeTypeConstructDescriptionDesigners
BBa_K1381011GeneratorJ23113-B0034-CheZThe chemotaxis controling gene CheZ with the promoter J23113Targeting Group
BBa_K1381012GeneratorJ23114-B0034-CheZThe chemotaxis controling gene CheZ with the promoter J23114Targeting Group
BBa_K1381013GeneratorJ23100-B0034-CheZThe chemotaxis controling gene CheZ with the promoter J23100Targeting Group
  • [1] Madigan MT, Martinko JM, Stahl DA, Clark DP. 2010. Brock biology of microorganisms. 13th ed. Benjamin Cummings, San Francisco.
  • [2] iGEM WITS CSIR 2011. 2011. Semi-solid agar motility assay. WWW document 21 September 2011: http://igem.org/wiki/images/a/a8/Motility_Assay_Protocol.pdf. Date visited 22 August 2014.
  • [3] iGEM SDU-Denmark 2010. 2010. Motility assay. WWW document 4 September 2014: http://2010.igem.org/Team:SDU-Denmark/K343004. Date visited 17 September 2014.
  • [4] Wolfe AJ, Berg HC. 1989. Migration of bacteria in semisolid agar. Proc Natl Acad Sci USA 86: 6973-6977.