Team:Goettingen/Team/Reporter
From 2013.igem.org
(→Discussion) |
(→Discussion) |
||
Line 122: | Line 122: | ||
===Discussion=== | ===Discussion=== | ||
- | Since we are in urgent need for novel antibiotics, we constructed two different c-di-AMP reporter systems which would allow the screening for compounds affecting c-di-AMP biofunction. One system was based on the c-di-AMP-binding transcriptional repressor DarR. In our system, DarR controlled the expression of the reporter gene GFP. E. coli cells transformed with a vector harboring the DarR reporter system exhibited no green fluorescence compared to cells transformed with a control plasmid lacking the DarR expression unit. This data indicated that DarR seems to be expressed and active as a repressor in E. coli. Even in the absence of c-di-AMP, DarR was likely to bind the DarR operator strongly and repressed GFP expression almost completely. Under these conditions, an intermediate GFP expression level would be more suitable for our purpose to screen for novel antibiotics. Such an intermediate expression level of GFP in the absence of c-di-AMP might be reached in two ways: For instance, one could try a promoter for DarR even weaker than the current one. A more successful approach might be directed mutagenesis of the DarR operator to reduce the binding affinity of DarR to this DNA sequence. | + | Since we are in urgent need for novel antibiotics, we constructed two different c-di-AMP reporter systems which would allow the screening for compounds affecting c-di-AMP biofunction. One system was based on the c-di-AMP-binding transcriptional repressor DarR. In our system, DarR controlled the expression of the reporter gene GFP. ''E. coli'' cells transformed with a vector harboring the DarR reporter system exhibited no green fluorescence compared to cells transformed with a control plasmid lacking the DarR expression unit. This data indicated that DarR seems to be expressed and active as a repressor in ''E. coli.'' Even in the absence of c-di-AMP, DarR was likely to bind the DarR operator strongly and repressed GFP expression almost completely. Under these conditions, an intermediate GFP expression level would be more suitable for our purpose to screen for novel antibiotics. Such an intermediate expression level of GFP in the absence of c-di-AMP might be reached in two ways: For instance, one could try a promoter for DarR even weaker than the current one. A more successful approach might be directed mutagenesis of the DarR operator to reduce the binding affinity of DarR to this DNA sequence. |
- | Regarding the alternative reporter system involving the c-di-AMP-responsive ''ydaO'' riboswitch, optimization is needed, as well. The results obtained from fluorescence microscopy showed that the riboswitch reporter construct is expressed in E. coli though the native ''Bacillus'' promoter and ''Bacillus'' RBS were employed. Hence, ''E. coli'' seems to be able to use these elements. The strong CFP fluorescence of the ''E. coli'' cells even suggests a strong promoter activity. The high transcript levels that might be caused by this strong promoter could account for the observed ineffectiveness of exogenously applied c-di-AMP: There might be more riboswitch than c-di-AMP to bind to. Consequently, one could try to increase the c-di-AMP amounts. Alternatively, one could fuse the riboswitch biobrick [http://parts.igem.org/Part:BBa_K1045005 BBA_K1045005] to a weaker promoter to reduce the mRNA levels. Another possibility might be to switch to low-copy plasmids for expression of the reporter system. If despite reduced transcript levels, c-di-AMP has no effect on the riboswitch reporter system, ''E. coli'' might be unable to take up c-di-AMP. Yet, this would not put an end to the construction of a screening system for antibiotics targeting c-di-AMP. It has been reported, that ''B. subtilis'' is able to take up c-di-AMP (Oppenheimer-Shaaman ''et al.'', 2011). Thus, one should put more effort in identifying the c-di-AMP importer from ''B. subtilis'' and probably also other bacteria. In parallel to this time-consuming way, one could try to synthesize c-di-AMP in vivo by expressing diadenylate cyclases in ''E. coli'' cells containing an optimized c-di-AMP reporter system. Using DACs of different activity, the output of the reporter system could be characterized. Finally, having a well characterized c-di-AMP reporter system combined with a DAC, it could possible to screen in vivo for antibiotics interfering not only with the function of the essential signaling nucleotide c-di-AMP, but also for its essential biosynthesis enzyme, the DAC. | + | Regarding the alternative reporter system involving the c-di-AMP-responsive ''ydaO'' riboswitch, optimization is needed, as well. The results obtained from fluorescence microscopy showed that the riboswitch reporter construct is expressed in ''E. coli'' though the native ''Bacillus'' promoter and ''Bacillus'' RBS were employed. Hence, ''E. coli'' seems to be able to use these elements. The strong CFP fluorescence of the ''E. coli'' cells even suggests a strong promoter activity. The high transcript levels that might be caused by this strong promoter could account for the observed ineffectiveness of exogenously applied c-di-AMP: There might be more riboswitch than c-di-AMP to bind to. Consequently, one could try to increase the c-di-AMP amounts. Alternatively, one could fuse the riboswitch biobrick [http://parts.igem.org/Part:BBa_K1045005 BBA_K1045005] to a weaker promoter to reduce the mRNA levels. Another possibility might be to switch to low-copy plasmids for expression of the reporter system. If despite reduced transcript levels, c-di-AMP has no effect on the riboswitch reporter system, ''E. coli'' might be unable to take up c-di-AMP. Yet, this would not put an end to the construction of a screening system for antibiotics targeting c-di-AMP. It has been reported, that ''B. subtilis'' is able to take up c-di-AMP (Oppenheimer-Shaaman ''et al.'', 2011). Thus, one should put more effort in identifying the c-di-AMP importer from ''B. subtilis'' and probably also other bacteria. In parallel to this time-consuming way, one could try to synthesize c-di-AMP in vivo by expressing diadenylate cyclases in ''E. coli'' cells containing an optimized c-di-AMP reporter system. Using DACs of different activity, the output of the reporter system could be characterized. Finally, having a well characterized c-di-AMP reporter system combined with a DAC, it could possible to screen in vivo for antibiotics interfering not only with the function of the essential signaling nucleotide c-di-AMP, but also for its essential biosynthesis enzyme, the DAC. |
<html> | <html> |
Revision as of 10:08, 1 October 2013