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Assembly Plan

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.

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 #, a selection of the results of our swarm plate assays is presented.

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

The following strains are present in the figure:

• 1) B0034_J23100-cheZ (UU2685)
• 2) B0034_J23113-cheZ (UU2685)
• 3) negative control (untransformed UU2685)
• 4) positive control (RP437)

In #A, #B and #C, strain no. 1 has been inoculated in the top section. In #D, #E and #F, 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 #, the positive controls are swarming in figure #A, #D, #E and #F. Swarming is visible as a cloud-like structure around the point where a strain was inoculated. For both #B and #C, the positive controls did not swarm.

In #D, besides the positive control, strain no. 2 is swarming. It should be noted that this was the only detected instance of swarming for fifteen swarm plate assays (three replicates of assays for five different strains).

Lastly, none of the negative controls swarmed in any of the swarm plate assays that were performed, and this is also visible in figure #.

The expected result of the swarm plate assay, the restoring of motility, was only observed once, and only in strain no. 2. As is illustrated by figure #A, #E and #F, constructs did frequently not swarm, while positive controls swarmed. The constructs did thus generally not succeed in restoring motility.

The scarceness of positive results on the swarm plate assay might also be caused by a lack of reproducibility in the methodology. This lack of reproducibility is illustrated by the fact that positive controls did not swarm on two swarm plate assays which are presented in figure #.

To assess the motility of the different strains in another way, the different strains were observed in a phase-contrast microscope. No particular difference in movement was observed however, between the wild-type (positive control), mutants (negative controls) and strains containing our constructs. All cells appeared to tumble, but there were no visible differences in the extent of tumbling.

Parts

• BBa_K1381011-J23113-B0034-CheZ
• BBa_K1381012-J23114-B0034-CheZ
• BBa_K1381013-J23100-B0034-CheZ

• [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: https://static.igem.org/mediawiki/igem.org/a/a8/Motility_Assay_Protocol.pdf. Date visited 22 August 2014.
• [3] iGEM SDU-Denmark 2010. 2010. Motility assay. WWW document 4 September 2014: https://2010.igem.org/Team:SDU-Denmark/K343004. Date visited 17 September 2014.