Difference between revisions of "Part:BBa K4244000"
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For the proper functioning of our biosensor construct we must consider that it needs to function in a colonic environment. The lldPRD promoter does not function in the presence of glucose or the absence of oxygen. Unfortunately, both conditions are found in the human colon, meaning that PlldPRD¬ cannot be used to sense lactate in a colonic environment (Schwerdtfeger et al., 2019). Zúñiga et al. developed a lactate sensitive promoter called ALPaGA (A Lactate Promoter Operating in Glucose and Anoxia) (Zúñiga et al., 2021). This promoter is sensitive to lactate in the presence of glucose and absence of oxygen, perfect for developing a biosensor to sense colon cancer. Because we want to use ALPaGA to sense lactate produced by cancer cells, we set out to characterize and compare the native lldPRD operon promoter to ALPaGA. This was done by analysing both promoters in two different conditions: 1) absence of glucose, presence of oxygen and 2) presence of glucose, absence of oxygen, the results of which can be seen in Figure 1 and 2.  
 
For the proper functioning of our biosensor construct we must consider that it needs to function in a colonic environment. The lldPRD promoter does not function in the presence of glucose or the absence of oxygen. Unfortunately, both conditions are found in the human colon, meaning that PlldPRD¬ cannot be used to sense lactate in a colonic environment (Schwerdtfeger et al., 2019). Zúñiga et al. developed a lactate sensitive promoter called ALPaGA (A Lactate Promoter Operating in Glucose and Anoxia) (Zúñiga et al., 2021). This promoter is sensitive to lactate in the presence of glucose and absence of oxygen, perfect for developing a biosensor to sense colon cancer. Because we want to use ALPaGA to sense lactate produced by cancer cells, we set out to characterize and compare the native lldPRD operon promoter to ALPaGA. This was done by analysing both promoters in two different conditions: 1) absence of glucose, presence of oxygen and 2) presence of glucose, absence of oxygen, the results of which can be seen in Figure 1 and 2.  
  
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[[File:Bba K4244000-ALPaGa-lldPRD.png|400px]|center]
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[[File:Bba K4244000-ALPaGa-lldPRD.png|400px|center]]
  
 
Figure 1 A dose response curve for Escherichia coli Nissle 1917 grown at microoxic conditions in M9 minimal media, supplemented with 22 mM glucose and L-lactate at different concentrations. Fluorescence was measured after 16h and corrected for OD600.
 
Figure 1 A dose response curve for Escherichia coli Nissle 1917 grown at microoxic conditions in M9 minimal media, supplemented with 22 mM glucose and L-lactate at different concentrations. Fluorescence was measured after 16h and corrected for OD600.

Revision as of 11:40, 5 October 2022


ALPaGA Lactate inducible promoter

For the proper functioning of our biosensor construct we must consider that it needs to function in a colonic environment. The lldPRD promoter does not function in the presence of glucose or the absence of oxygen. Unfortunately, both conditions are found in the human colon, meaning that PlldPRD¬ cannot be used to sense lactate in a colonic environment (Schwerdtfeger et al., 2019). Zúñiga et al. developed a lactate sensitive promoter called ALPaGA (A Lactate Promoter Operating in Glucose and Anoxia) (Zúñiga et al., 2021). This promoter is sensitive to lactate in the presence of glucose and absence of oxygen, perfect for developing a biosensor to sense colon cancer. Because we want to use ALPaGA to sense lactate produced by cancer cells, we set out to characterize and compare the native lldPRD operon promoter to ALPaGA. This was done by analysing both promoters in two different conditions: 1) absence of glucose, presence of oxygen and 2) presence of glucose, absence of oxygen, the results of which can be seen in Figure 1 and 2.

Bba K4244000-ALPaGa-lldPRD.png

Figure 1 A dose response curve for Escherichia coli Nissle 1917 grown at microoxic conditions in M9 minimal media, supplemented with 22 mM glucose and L-lactate at different concentrations. Fluorescence was measured after 16h and corrected for OD600.


From this data it can be concluded that ALPaGA performs similarly to the native lldPRD operon promoter in conditions without glucose and with oxygen, but greatly outperforms the native promoter when there is absence of oxygen and presence of glucose. This means that this promoter is more viable to sense lactate in colonic environments. Furthermore, it can be argued that the ALPaGA promoter more efficiently sense tumours in general by means of lactate, since the cancer microenvironment contains little oxygen but still contains glucose (Reinfeld et al., 2021). Therefore, we present the improved version of the lldPRD operon promoter into the iGEM registry, ALPaGA as BBa_K4244000. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



References

Reinfeld, et al.,(2021). Cell-programmed nutrient partitioning in the tumour microenvironment. Nature, 593(7858), 282–288. https://doi.org/10.1038/s41586-021-03442-1

Schwerdtfeger, L. A., Nealon, N. J., Ryan, E. P., & Tobet, S. A. (2019). Human colon function ex vivo: Dependence on oxygen and sensitivity to antibiotic. PLOS ONE, 14(5), e0217170. https://doi.org/10.1371/JOURNAL.PONE.0217170

Zúñiga, A., Camacho, M., Chang, H. J., Fristot, E., Mayonove, P., Hani, E. H., & Bonnet, J. (2021). Engineered l-Lactate Responding Promoter System Operating in Glucose-Rich and Anoxic Environments. ACS Synthetic Biology, 10(12), 3527–3536. https://doi.org/10.1021/acssynbio.1c00456