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Team:Imperial College London/Extras/Collaboration
From 2011.igem.org
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<h1>Collaboration</h1> | <h1>Collaboration</h1> | ||
| - | <p>The Imperial College London team collaborated with WITS CSIR iGEM team. Aspects that were similar in both projects included rewiring of the chemotaxis <i>E. coli</i>, however the design was different for both projects. WE have tried to rewire chemotaxis of <i>E. coli</i> using structurally similar chemoreceptors that then use endogenous molecular mechanism of chemotaxis, whereas WITS - CSIR team used novel concept of riboswitches to modify the functioning of the chemotaxis pathway in <i>E. coli</i>. Collaboration was performed in both wetlab and modelling aspects. </p> | + | <p>The Imperial College London team collaborated with WITS-CSIR iGEM team. Aspects that were similar in both projects included rewiring of the chemotaxis <i>E. coli</i>, however the design was different for both projects. WE have tried to rewire chemotaxis of <i>E. coli</i> using structurally similar chemoreceptors that then use endogenous molecular mechanism of chemotaxis, whereas WITS-CSIR team used novel concept of riboswitches to modify the functioning of the chemotaxis pathway in <i>E. coli</i>. Collaboration was performed in both wetlab and modelling aspects. </p> |
<p><h2>Wet Lab collaboration</h2></p> | <p><h2>Wet Lab collaboration</h2></p> | ||
| - | <p> 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 occured and a number of relevant scientific papers were exchanged, that have given important clues for both teams 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, nor did they characterise one of our biobricks. Our dry lab team however, have modelled part of their design.</p> | + | <p> 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 occured and a number of relevant scientific papers were exchanged, that have given important clues for both teams 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, nor did they characterise one of our biobricks. Our dry lab team however, have modelled part of their design.</p> |
<h3> Know - How exchange</h3> | <h3> Know - How exchange</h3> | ||
| - | <p>Both teams have used qualitative assays to analyze chemotactic movement. Even though the set up of the assay used by each team was a bit different the basics stayed the same. Both teams have discussed, what media 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 is sufficient. Both teams have agreed that growing overnight culture in LB does not affect bacterial ability to swim. Another aspect of qualitative assays is semi - solid agar. Both teams have used semi solid agar based on M9 minimal media. Another factor during chemotaxis is visualisation, WITS - CSIR 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 team have suggested to use TTC, a chemical that can turn red while being metabolised by cells. | + | <p>Both teams have used qualitative assays to analyze chemotactic movement. Even though the set up of the assay used by each team was a bit different the basics stayed the same. Both teams have discussed, what media 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 is sufficient. Both teams have agreed that growing overnight culture in LB does not affect bacterial ability to swim. Another aspect of qualitative assays is semi - solid agar. Both teams have used semi solid agar based on M9 minimal media. Another factor during chemotaxis is visualisation, WITS-CSIR 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 team have suggested to use TTC, a chemical that can turn red while being metabolised by cells. |
In terms of quantitative assays, we have suggested to WITS - CSIR team to combine commonly used capillary assay for testing of chemotactic movements with the subsequent data collection using flow cytometer as oppose to method using CFU for quantification of bacterial chemotaxis. </p> | In terms of quantitative assays, we have suggested to WITS - CSIR team to combine commonly used capillary assay for testing of chemotactic movements with the subsequent data collection using flow cytometer as oppose to method using CFU for quantification of bacterial chemotaxis. </p> | ||
| - | <p><h2> | + | <p><h2>Dry Lab collaboration</h2></p> |
| - | + | <p>Regarding to modeling, team from Imperial College London had several skype meetings with WITS-CSIR iGEM team to discuss the design of chemotaxis modelling, and theories behind our models. In order to share our modelling ideas and exhchange some of our models, we modelled the chemotaxis pathway for WITS-CSIR team. The modeling results are shown below. </p> | |
| - | + | <p><h3> MODELLING RESULTS</h3></p> | |
| - | <p> | + | <p>WITS-CSIR iGEM team intends to use 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. WITS-CSIR 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]. </p> |
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| - | <p> The theophylline | + | <p> The theophylline riboswitch can be modeled in three differential equations (Equation 1). [2] </p> |
<p> <img src="https://static.igem.org/mediawiki/2011/a/a1/Cequ1.png" /> | <p> <img src="https://static.igem.org/mediawiki/2011/a/a1/Cequ1.png" /> | ||
<p> 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. </p> | <p> 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. </p> | ||
Revision as of 11:59, 15 September 2011
Collaboration
The Imperial College London team collaborated with WITS-CSIR iGEM team. Aspects that were similar in both projects included rewiring of the chemotaxis 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 endogenous molecular mechanism of chemotaxis, whereas WITS-CSIR team used novel concept of riboswitches to modify the functioning of the chemotaxis pathway in E. coli. Collaboration was performed in both wetlab and modelling aspects.
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 occured and a number of relevant scientific papers were exchanged, that have given important clues for both teams 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, nor did they characterise one of our biobricks. Our dry lab team however, have modelled part of their design.
Know - How exchange
Both teams have used qualitative assays to analyze chemotactic movement. Even though the set up of the assay used by each team was a bit different the basics stayed the same. Both teams have discussed, what media 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 is sufficient. Both teams have agreed that growing overnight culture in LB does not affect bacterial ability to swim. Another aspect of qualitative assays is semi - solid agar. Both teams have used semi solid agar based on M9 minimal media. Another factor during chemotaxis is visualisation, WITS-CSIR 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 team have suggested to use TTC, a chemical that can turn red while being metabolised by cells. In terms of quantitative assays, we have suggested to WITS - CSIR team to combine commonly used capillary assay for testing of chemotactic movements with the subsequent data collection using flow cytometer as oppose to method using CFU for quantification of bacterial chemotaxis.
Dry Lab collaboration
Regarding to modeling, team from Imperial College London had several skype meetings with WITS-CSIR iGEM team to discuss the design of chemotaxis modelling, and theories behind our models. In order to share our modelling ideas and exhchange some of our models, we modelled the chemotaxis pathway for WITS-CSIR team. The modeling results are shown below.
MODELLING RESULTS
WITS-CSIR iGEM team intends to use 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. WITS-CSIR 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 modeled 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 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(a) and Fig.1(b) below. In addition, the response curve of CheZ against theophylline concentration with different theophylline transport rate was illustrated in Fig.1(c).
Fig.1(a) Expression of CheZ VS time
Fig.1(b) Intracellular Theophyline concentration VS time
Fig.1(c) Response curve of CheZ concentration VS intracellular concentration
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 shown 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.
II. PARAMETERS
III. REFERENCE
[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.’ PLoS Comput Biol 5(4): e1000363. doi:10.1371/journal.pcbi.1000363 Parameters:
