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Revision as of 18:26, 21 September 2011

Collaboration

The Imperial College London team collaborated with the WITS-CSIR_SA iGEM team. Aspects that were similar in both projects included rewiring of chemotaxis in E. coli, however the design was different for both projects. We have tried to rewire chemotaxis of E. coli using structurally similar chemoreceptors that then use the endogenous molecular mechanism of chemotaxis, whereas the WITS-CSIR_SA team used a novel concept of riboswitches to modify the functioning of the chemotaxis pathway in E. coli. We collaborated in both wetlab and modelling aspects.

1. Wet lab collaboration

Chemotaxis assays are quite difficult to perform correctly and therefore know-how, tips and tricks are essential to know in order to get chemotaxis assays working. In sight of this, a number of skype conversations occurred and a number of relevant scientific papers were exchanged that have given important clues for both teams on how to proceed with the testing for chemotaxis. Due to the distance, short time frame and different BioBricks used to rewire chemotaxis, we did not characterise a BioBrick of WITS-CSIR_SA, nor did they characterise one of our BioBricks. Our dry lab team however, have modelled part of their design.

Figure 1. The members of the WITS-CSIR_SA team

1.1 Know-how exchange

Both teams have used qualitative assays to analyse chemotactic movement. Even though the setup of the assays used by each team were slightly different, the basic principles remained the same. Both teams have discussed the type of media that should be used to grow cells, as some sources claim that it is necessary for chemotaxis to have overnight culture grown in Tryptone broth, whereas other sources say that overnight growth in LB broth is sufficient. Both teams have agreed that growing overnight culture in LB does not affect the ability of the bacteria to swim.

Another aspect of qualitative assays is semi-solid agar. Both teams have used semi-solid agar based on M9 minimal medium. Another factor during chemotaxis is visualisation, WITS-CSIR_SA team have added GFP into their construct, so visualisation of their qualitative assay was easy, however since our construct does not have GFP, visualisation would be more difficult. WITS-CSIR_SA team have suggested to use Tetrazolium Chloride, a chemical that is metabolised by cells to produce a red pigment, and is often used as an indicator of cellular respiration.

In terms of quantitative assays, we have suggested to WITS-CSIR_SA team that they combine a commonly-used capillary assay for testing of chemotactic movement with the subsequent data collection using a flow cytometer as opposed to using colony forming units for quantification of bacterial populations.

2. Dry Lab collaboration

The modellers of the Imperial College London team had several Skype meetings with WITS-CSIR iGEM team to discuss the design of the chemotaxis modelling, and the theories behind our models. In order to share our modelling ideas and exchange some of our models, we modelled the theophylline riboswitch pathway for WITS-CSIR_SA team. The modeling results are shown below.

2.1 Modelling results

The WITS-CSIR_SA 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, and WITS-CSIR_SA have used a theophylline riboswitch to control the expression of CheZ in CheZ mutants in order to engineer the bacterial movement towards theophylline[1]. They are 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 cannot occur. In the presence of theophylline, the shape of the aptamer (riboswitch) changes and the start codon is exposed[1]. Thus, the higher the concentration of the theophylline, the more will enter the cell and the higher the expression of CheZ and the higher the level of directed movement[1]. The theophylline riboswitch can be modelled in three differential equations (Equation 1)[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 at the transcription and translation levels respectively (Equation 2 below [2]). K_Φ is the equilibrium constant at transcriptional level and K_Θ is the equilibrium constant at translation level [2].

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 Figure 3(a) and Figure 3(b) below. In addition, the response curve of CheZ against theophylline concentration with different theophylline transport rate was illustrated in Figure 3(c).

Figure 2. Expression of CheZ vs. timeThe expression of CheZ increases with increase of transport rate. With higher theophyline transport rate bacteria will produce more CheZ and therefore a higher level of directed movement.

Figure 3. Intracellular theophylline vs. time The intracellular theophylline level increases with transport rate. It means we can enhance CheZ expression with increasing theophylline chemoreceptors.

Figure 4. Response curve of CheZ vs. intracellular theophylline. The CheZ response curve shows that the transport rate can be used to tune CheZ response, the threshold intracellular concentration of theophylline required to trigger CheZ response increases with transport rate.

In conclusion, from modelling we know that the concentration of CheZ and intracellular theophylline increase with increasing theophylline transport rate. Also, the expression of CheZ can be tuned by this transport rate. Therefore, it is believed that the CheZ level and the level of directed movement of bacteria can be increased by expressing more theophylline transporter in the cell membrane.

2.2 Parameters

2.3 Matlab code

2.4 References

[1] Topp S, Gallivan JP (2007) Guiding bacteria with small molecules and RNA. J Am Chem Soc 129(21): 6807-6811.

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

[3] Beisel CL and Smolke CD (2009) Design principles for riboswitch function. PLoS Comput Biol 5(4): e1000363, doi:10.1371/journal.pcbi.1000363.