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Team:Uppsala/Project Targeting
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The Bacticciles is in its Targeting mode, expressing alot of cheZ and sRNA, spot42_USP45. |  |
Background
Movement and Chemotaxis
Chemotaxis is the cell’s 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 an immotile bacterium. 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 Sensing) 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 cheZ (gene) combined with the regulatory system designed by the Sensing group. 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 [2].
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 the regulatory system designed by the Sensing group 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.
The Bacticciles is in its Targeting mode, expressing alot of cheZ and sRNA, spot42_USP45. |  ResultAll 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.
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:
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.
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:
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.
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