The Wits CSIR iGEM team collaborated with Imperial College London, both for the modelling and the potential suitability of motility experiments in the testing of our machines. The biologists in our team and those at Imperial College London had a number of skype conferences to discuss the progress of our lab work as well as to share insight into both qualitative and quantitative assay for motility.

We provided Imperial College London with some ideas regarding motility assays using TTC in stab agar. Their team gave us some tips about the preparation of cells for motility experiments and recommended spinning down the bacteria and resuspending them in a low volume of broth to increase the density of cells. They also recommended the use of capillary motility assays. There was an exchange of protocols both ways to aid in the set-up of our experiments. A week later we discussed the progress we had made in the lab. We gave each other tips and suggested more assays to indicate the ability of our bacteria to move towards a stimulus. Imperial College London shared with us a type of capillary assay they were performing at the time and we, too, shared a protocol with them which is detailed in Gullivan and Topps (2006) paper, Guiding bacteria with small molecules and RNA.

Collaboration was also performed between the engineers to improve the modeling for both the teams. Several Skype conferences were held between the team from Imperial College London and the Wits CSIR team to discuss the design and theory behind our own chemotaxis models. The collaborative effort resulted in the Imperial College London kindly providing us with a model for our theophylline riboswitches. Their modelling results are as follows:

The Wits CSIR iGEM team intend to use a riboswitch to reprogram the chemotactic behavior of E.coli. The project includes engineering the attraction of the bacteria to theophylline[1]. CheZ is an important protein controlling the chemotaxis of bacteria. They used a theophylline riboswitch to control the expression of CheZ in CheZ deletion mutants in order to engineer the bacteria's movement towards theophylline[1]. We're using a riboswitch sensitive to theophylline to control the expression of CheZ. In the absence of theophylline, the start codon is covered so the translation of the strand cannot occur. In the presence of theophylline, the conformation of the riboswitch changes and the ribosome binding site is exposed[1]. Thus, the higher the concentration of the theophylline, the more will enter the cell resulting in the up regulation of CheZ expression. This will increase the frequency of directed movement[1].

The theophylline riboswitch can be modeled in three differential equations [2].

M, CheZ and T respectively stands for the concentration of CheZ mRNA, the concentration of protein CheZ and the concentration of theophylline. The constants α,β,γ,ξ and δ are all positive, and respectively denote the CheZ-promoter transcription rate, the CheZ-mRNA translation rate and the mRNA, CheZ and theophylline degradation-plus-dilution-rates. ζ(Text) is the theophylline transport rate per unit CheZ concentration. It is a function of number of theophylline receptors and external theophylline concentration. The function ϕ(T) and Θ(T) denote the theophylline-governed regulation ay the transcriptional and translation levels respectively (equation below [2]). KΦ is the equilibrium constant at transcriptional level and KΘ is the equilibrium constant at translation level.

Varying the parameter ζ(Text) of above model could help us to understand how the number of receptors and external theophylline concentration effect the intracellular concentration of theothyline and hence the expression level of CheZ. The results are shown in Fig 1 and Fig 2 below. In addition, the response curve of CheZ against theophylline concentration with different theophylline transport rate was illustrated in Fig 3.

Both the expression of CheZ and intracellular concentration increases with increase of transport rate. The bacteria will produce more CheZ and therefore higher level of directed movement. The CheZ response curve shows that the transport rate can be used to tune the CheZ response, as the threshold intracellular concentration of theophylline required to trigger the CheZ response increases with transport rate.

References

[1] S. Topp , Justin P. Gallivan(2007) ‘Guiding bacteria with small molecules and RNA’, J. AM. CHEM. SOC. 2007, 129,6807-6811. VOL. 129, NO. 21,2007

[2] M. Santillan , M. C. Mackey (2005)’Dynamic behavior of the B12 riboswitch’ Phys. Biol 2(2005) 29-35. Doi: 10.1088/1478-3967/2/1/004

[3] Beisel CL, Smolke CD (2009) ‘Design Principles for Riboswitch Function.’PLoSComputBiol 5(4): e1000363. doi:10.1371/journal.pcbi.1000363