Team:ETH Zurich/expresults/qs

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Revision as of 20:57, 15 October 2014

Quorum Sensing

For our Mosiacoli project, we were looking for molecular systems that allow orthogonal cell-to-cell communication in order to implement connected XOR logic gates (ref project overview). As described above and elsewhere (ref to biotools) we decided to exploit the quorum-sensing systems LuxI/LuxR, LasI/LasR, and RhlI/RhlR (ref to qs parts) in order to achieve the required orthogonal cell-to-cell communication. Even though the corresponding inducer molecules are commercially available and the systems often used (ref ref), in particular in iGEM projects (ref ref), potential cross-talk activity between the different systems may be a severe problem 9ref). In order to address that challenge, we have investigated the possible cross-talk on several molecular levels: a) unspecific binding of inducer molecules to one specific regulator (table 1), b) unspecific binding of regulator molecules to one specific promoter sequence (table 2), and c) combinations of both.

PUT TABLE HERE

In the laboratory, we measured a) a given promoter with its corresponding regulator and a different inducer molecule, b) a given promoter with an unspecific regulator and a particular inducer, and c) a given promoter with both regulator and inducer being unspecific. This gives in total 27 possible combinations (ref to sequences). The output was assessed via sfGFP and measured in terms of fluorescence on microtiter-plate scale. In addition, we assessed both the unregulated and regulated versions of the quorum sensing modules (see above ref).

PUT MATRICES/GRAPHS HERE

As indicated in the graphs above, we found and quantitatively characterized all three levels of cross-talk stated above. Unspecific inducer binding to the regulators as well as unspecific binding of the regulator to the promoter occurred in all possible combinations. To conclude, we were not able to find an orthogonal quorum-sensing pair due to inevitable cross-talk.

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 == Quorum Sensing ==
-For our project, we were looking for molecular systems that allow orthogonal cell-to-cell communication to implement connected XOR logic gates (ref project overview). As described above and elsewhere (ref to biotools) we decided to exploited the quorum-sensing systems LuxI/LuxR, LasI/LasR, and RhlI/RhlR (ref to qs parts) in order to achieve this orthogonal cell-to-cell communication. Even though the corresponding inducer molecules are commercially available  and the systems often used, in particular in iGEM projects (ref ref), potential cross-talk activity between the different systems may be a severe problem. In order to address that challenge, we have investigated the possible cross-talk on several molecular levels: a) unspecific binding of inducer molecules to one specific regulator (table 1), b) unspecific binding of regulator molecules to one specific promoter sequence (table 2), and c) combinations of both.
+For our Mosia''coli'' project, we were looking for molecular systems that allow orthogonal cell-to-cell communication in order to implement connected XOR logic gates (ref project overview). As described above and elsewhere (ref to biotools) we decided to exploit the quorum-sensing systems LuxI/LuxR, LasI/LasR, and RhlI/RhlR (ref to qs parts) in order to achieve the required orthogonal cell-to-cell communication. Even though the corresponding inducer molecules are commercially available and the systems often used (ref ref), in particular in iGEM projects (ref ref), potential cross-talk activity between the different systems may be a severe problem 9ref). In order to address that challenge, we have investigated the possible cross-talk on several molecular levels: a) unspecific binding of inducer molecules to one specific regulator (table 1), b) unspecific binding of regulator molecules to one specific promoter sequence (table 2), and c) combinations of both.
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-In the laboratory, we measured a) a given promoter with its corresponding regulator and a different inducer molecule, b) a given promoter with an unspecific regulator and a particular inducer, and c) a given promoter with both regulator and inducer being unspecific. This gives in total 27 possible combinations. The output was again assessed via sfGFP and measured in terms of fluorescence on microtiter-plate scale. In addition, we assessed both the 'normal' and regulated versions of the quorum sensing modules (see above ref).
+In the laboratory, we measured a) a given promoter with its corresponding regulator and a different inducer molecule, b) a given promoter with an unspecific regulator and a particular inducer, and c) a given promoter with both regulator and inducer being unspecific. This gives in total 27 possible combinations (ref to sequences). The output was assessed via sfGFP and measured in terms of fluorescence on microtiter-plate scale. In addition, we assessed both the unregulated and regulated versions of the quorum sensing modules (see above ref).