Difference between revisions of "Part:BBa K4244000"

Revision as of 11:42, 11 October 2022

ALPaGA Lactate inducible promoter

Motivation

The goal of our project is to develop a self-test for colorectal cancer. A general cancer biomarker we want to sense to achieve this is lactate (1). Some bacteria, of which Escherichia coli contain an operon called lldPRD, which is responsible for the dissimilation of lactate. This operon can respond to lactate due to its transcription factor LldR which binds to the operons promoter, PlldPRD BBa_K822000. If lactate is absent the LldR represses the operon, however, if lactate is present LldR activates expression of the operon (2). This property has been used to produce genetic elements that are sensitive to the presence of lactate and applied as a basis for whole-cell lactate biosensors (3). In our projec,t we also aim to apply this to our bacterial chassis E. coli Nissle 1917 to develop a lactate-sensitive biosensor.
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 (4). Zúñiga et al. developed a lactate sensitive promoter called ALPaGA (A Lactate Promoter Operating in Glucose and Anoxia) (5). This promoter is sensitive to lactate in the presence of glucose and the 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 Figures 1 and 2.

Results

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Figure 1: A dose-response curve for Escherichia coli Nissle 1917 grown aerobically in M9 minimal media, supplemented with 15 mM succinate and L-lactate at different concentrations. Fluorescence was measured after 16h and corrected for OD600.

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Figure 2: 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 figure 1 can be seen that PlldPRD and ALPaGA perform similarly when glucose is absent, and oxygen is present. However, when simulating colonic conditions, meaning the presence of glucose, and the absence of oxygen, the fluorescence output of the ALPaGA promoter is higher at all lactate concentrations compared to lldPRD.

Conclusion

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 (6). Therefore, we present the improved version of the lldPRD operon promoter into the iGEM registry, ALPaGA.

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

1. Liberti M v., Locasale JW. The Warburg Effect: How Does it Benefit Cancer Cells? Trends Biochem Sci. 2016 Mar 1;41(3):211–8.
2. Aguilera L, Campos E, Giménez R, Badía J, Aguilar J, Baldoma L. Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli. J Bacteriol. 2008;190(8):2997–3005.
3. Goers L, Ainsworth C, Goey CH, Kontoravdi C, Freemont PS, Polizzi KM. Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production. Biotechnol Bioeng [Internet]. 2017 Jun 1 [cited 2022 Apr 12];114(6):1290–300. Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/bit.26254
4. Schwerdtfeger LA, Nealon NJ, Ryan EP, Tobet SA. Human colon function ex vivo: Dependence on oxygen and sensitivity to antibiotic. PLoS One [Internet]. 2019 May 1 [cited 2022 Sep 30];14(5):e0217170. Available from: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0217170
5. Zúñiga A, Camacho M, Chang HJ, Fristot E, Mayonove P, Hani EH, et al. Engineered l-Lactate Responding Promoter System Operating in Glucose-Rich and Anoxic Environments. ACS Synth Biol [Internet]. 2021;10(12):3527–36. Available from: https://doi.org/10.1021/acssynbio.1c00456
6. Reinfeld BI, Madden MZ, Wolf MM, Chytil A, Bader JE, Patterson AR, et al. Cell-programmed nutrient partitioning in the tumour microenvironment. Nature. 2021 May 13;593(7858):282–8.

@@ Line 6: / Line 6: @@
-The goal of our project is to develop a '''self-test''' for colorectal cancer. A general cancer biomarker we want to sense to achieve this is lactate (1). Some bacteria, of which Escherichia coli contain an operon called lldPRD, which is responsible for the dissimilation of lactate. This operon can respond to lactate due to its transcription factor LldR which binds to the operons promoter, PlldPRD <partinfo>BBa_K822000</partinfo>. If lactate is absent the LldR represses the operon, however if lactate is present LldR activates expression of the operon (2). This property has been used to produce genetic elements that are sensitive to the presence of lactate and applied as a basis for whole-cell lactate biosensors (3). In our project we also aim to apply this to our bacterial chassis E. coli Nissle 1917 to develop a lactate sensitive biosensor.<br>
+The goal of our project is to develop a '''self-test''' for colorectal cancer. A general cancer biomarker we want to sense to achieve this is lactate (1). Some bacteria, of which Escherichia coli contain an operon called lldPRD, which is responsible for the dissimilation of lactate. This operon can respond to lactate due to its transcription factor LldR which binds to the operons promoter, PlldPRD <partinfo>BBa_K822000</partinfo>. If lactate is absent the LldR represses the operon, however, if lactate is present LldR activates expression of the operon (2). This property has been used to produce genetic elements that are sensitive to the presence of lactate and applied as a basis for whole-cell lactate biosensors (3). In our projec,t we also aim to apply this to our bacterial chassis E. coli Nissle 1917 to develop a lactate-sensitive biosensor.<br>
-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 (4). Zúñiga et al. developed a lactate sensitive promoter called ALPaGA (A Lactate Promoter Operating in Glucose and Anoxia) (5). 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 (4). Zúñiga et al. developed a lactate sensitive promoter called ALPaGA (A Lactate Promoter Operating in Glucose and Anoxia) (5). This promoter is sensitive to lactate in the presence of glucose and the 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 Figures 1 and 2.
 ===== Results=====
-[[File:Bba_K4244000-ALPaGa-lldPRD-microoxic.svg|thumb|500px|center|<font size="1">Figure 1: A dose response curve for Escherichia coli Nissle 1917 grown aerobically in M9 minimal media, supplemented with 15 mM succinate and L-lactate at different concentrations. Fluorescence was measured after 16h and corrected for OD600. </font>]]
+[[File:Bba_K4244000-ALPaGa-lldPRD-microoxic.svg|thumb|500px|center|<font size="1">Figure 1: A dose-response curve for Escherichia coli Nissle 1917 grown aerobically in M9 minimal media, supplemented with 15 mM succinate and L-lactate at different concentrations. Fluorescence was measured after 16h and corrected for OD600. </font>]]
 [[File:Bba_K4244000-ALPaGa-lldPRD-aerobic.svg|thumb|500px|center|<font size="1">Figure 2: 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. </font>]]
-From figure 1 can be seen that PlldPRD and ALPaGA perform similarly when glucose is absent, and oxygen is present. However, when simulating colonic conditions, meaning presence of glucose, and absence of oxygen, the fluorescence output of the ALPaGA promoter is higher at all lactate concentrations compared to lldPRD.
+From figure 1 can be seen that PlldPRD and ALPaGA perform similarly when glucose is absent, and oxygen is present. However, when simulating colonic conditions, meaning the presence of glucose, and the absence of oxygen, the fluorescence output of the ALPaGA promoter is higher at all lactate concentrations compared to lldPRD.