Difference between revisions of "Part:BBa K3338008"

 
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<partinfo>BBa_K3338008 short</partinfo>
 
<partinfo>BBa_K3338008 short</partinfo>
  
Long description
 
 
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===Usage and Biology===
 
===Usage and Biology===
  
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The human interleukin-6 promoter originally controls the expression of the cytokine Il-6 that is involved in the regulation of the acute-phase response to injury and infection but also other processes like hematopoiesis and embryonal development (Heinrich <i>el al.</i> 2003). The regulation of transcription exerted by the IL-6 promoter is mainly controlled by several cis-acting response elements present within the promoter region including binding motifs for NF-&#954;B, NF-IL6, CREB, C/EBP, AP-1 and AP-2 (Xiao <i>et al.</i> 2004, Beetz <i>et al.</i> 2000). The regulation of the IL-6 promoter depends on the cell type and the stimulus. This means that different transcription factors are needed under different conditions. In our study we used LPS to induce IL-6 promoter activation. In this case the transcription factors NF-&#954;B and AP1 play important roles (Xiao <i>et al.</i> 2004, Liu <i>et al.</i> 2018). The activation of the promoter is achieved by the cooperative binding of NF-&#954;B and c-Jun (AP-1) (Xiao <i>et al.</i> 2004). AP-1- and NF-&#954;B-translocation to the nucleus is triggered downstream of Toll like receptor (TLR) signaling cascades involving TRIF, MyD88, RIPK1 and TAK1. Toll like receptors are activated by a variety of pathogen-associated molecular patterns (Kawai and Akira 2007).
<span class='h3bb'>Sequence and Features</span>
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In our study we used it as a LPS sensor for our inflammatory toxin sensor. After treatment of transfected HeLa cells with LPS a significant and long-lasting upregulation of the promoter activity was observed. However, it does not match the Biobrick assembly standard requirements. Therefore, we generated a mutant that is Biobrick compatible (<html><a href="https://parts.igem.org/Part:BBa_K3338005">BBa_K3338005</a></html>) but unfortunately it does not show LPS sensitivity.
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===Sequence and Features===
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<partinfo>BBa_K3338008 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3338008 SequenceAndFeatures</partinfo>
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=Characterization=
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In order to characterize the human wildtype interleukin-6 promoter we tested its activity following treatment of HeLa cells with different LPS-concentrations using an IL-6 P-hGLuc construct. The results in figure 1 indicate that IL-6 promoter activity significantly increases when the cells are treated with rising amounts LPS.
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<img src="https://2020.igem.org/wiki/images/d/de/T--Hannover--results_promoter_IL6.png" class="center">
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Figure 2: Relative activity of IL6 promoter in HeLa cells 24 hours (A) and 48 hours (B) after treatment with different concentrations of LPS for 3 hours, normalized to untreated control (0 µg/mL LPS). Data shown represents mean ± SEM of n=4 biological replicates. Statistical analysis was performed by unpaired t-test in comparison to untreated control, significance level: 10 %, significance is indicated by asterisk.
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We also assed the basal promoter activity and compared it to the CMV promoter activity (see figure 2). The IL-6 promoter shows a comparable basal activity with the CMV promoter. This behavior is surprising but can be possibly explained by differences in transfection efficiencies of both promoter constructs. However, further experiments are needed to clarify.
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<img src="https://2020.igem.org/wiki/images/9/93/T--Hannover--results_promoter_basal.png" class="center">
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Figure 2: Relative basal activity of all tested promoters in HeLa cells 48 hours (A) and 72 hours (B) after transfection. Data was normalized to CMV promoter which served as reference. Data shown represents mean ± SEM of n=4 biological replicates.
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===Summary===
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In summing up, the interleukin-6 promoter exhibits a significant LPS-sensitivity and a low basal activity making it ideal for use as a LPS (inflammatory toxin-) sensor.
  
  
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<partinfo>BBa_K3338008 parameters</partinfo>
 
<partinfo>BBa_K3338008 parameters</partinfo>
 
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=References=
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Beetz, A., Peter, R. U., Oppel, T., Kaffenberger, W., Rupec, R. A., Meyer, M., van Beuningen, D., Kind, P., & Messer, G. (2000). NF-kappaB and AP-1 are responsible for inducibility of the IL-6 promoter by ionizing radiation in HeLa cells. <i>International journal of radiation biology</i>, 76(11), 1443–1453.
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Heinrich, P. C., Behrmann, I., Haan, S., Hermanns, H. M., Müller-Newen, G., & Schaper, F. (2003). Principles of interleukin (IL)-6-type cytokine signalling and its regulation. <i>The Biochemical journal</i>, 374(Pt 1), 1–20.
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Kawai, T., & Akira, S. (2007). Signaling to NF-kappaB by Toll-like receptors. <i>Trends in molecular medicine</i>, 13(11), 460–469.
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Liu, X., Yin, S., Chen, Y., Wu, Y., Zheng, W., Dong, H., Bai, Y., Qin, Y., Li, J., Feng, S., & Zhao, P. (2018). LPS‑induced proinflammatory cytokine expression in human airway epithelial cells and macrophages via NF‑κB, STAT3 or AP‑1 activation. <i>Molecular medicine reports</i>, 17(4), 5484–5491.
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Xiao, W., Hodge, D. R., Wang, L., Yang, X., Zhang, X., & Farrar, W. L. (2004). NF-kappaB activates IL-6 expression through cooperation with c-Jun and IL6-AP1 site, but is independent of its IL6-NFkappaB regulatory site in autocrine human multiple myeloma cells. <i>Cancer biology & therapy</i>, 3(10), 1007–1017.

Latest revision as of 18:23, 27 October 2020


Interleukin-6 Promoter (IL-6 P)

Usage and Biology

The human interleukin-6 promoter originally controls the expression of the cytokine Il-6 that is involved in the regulation of the acute-phase response to injury and infection but also other processes like hematopoiesis and embryonal development (Heinrich el al. 2003). The regulation of transcription exerted by the IL-6 promoter is mainly controlled by several cis-acting response elements present within the promoter region including binding motifs for NF-κB, NF-IL6, CREB, C/EBP, AP-1 and AP-2 (Xiao et al. 2004, Beetz et al. 2000). The regulation of the IL-6 promoter depends on the cell type and the stimulus. This means that different transcription factors are needed under different conditions. In our study we used LPS to induce IL-6 promoter activation. In this case the transcription factors NF-κB and AP1 play important roles (Xiao et al. 2004, Liu et al. 2018). The activation of the promoter is achieved by the cooperative binding of NF-κB and c-Jun (AP-1) (Xiao et al. 2004). AP-1- and NF-κB-translocation to the nucleus is triggered downstream of Toll like receptor (TLR) signaling cascades involving TRIF, MyD88, RIPK1 and TAK1. Toll like receptors are activated by a variety of pathogen-associated molecular patterns (Kawai and Akira 2007).

In our study we used it as a LPS sensor for our inflammatory toxin sensor. After treatment of transfected HeLa cells with LPS a significant and long-lasting upregulation of the promoter activity was observed. However, it does not match the Biobrick assembly standard requirements. Therefore, we generated a mutant that is Biobrick compatible (BBa_K3338005) but unfortunately it does not show LPS sensitivity.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 369
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1347
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 220
    Illegal BamHI site found at 395
    Illegal XhoI site found at 1581
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 369
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 369
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 340
    Illegal BsaI.rc site found at 387
    Illegal BsaI.rc site found at 696


Characterization

In order to characterize the human wildtype interleukin-6 promoter we tested its activity following treatment of HeLa cells with different LPS-concentrations using an IL-6 P-hGLuc construct. The results in figure 1 indicate that IL-6 promoter activity significantly increases when the cells are treated with rising amounts LPS.


Figure 2: Relative activity of IL6 promoter in HeLa cells 24 hours (A) and 48 hours (B) after treatment with different concentrations of LPS for 3 hours, normalized to untreated control (0 µg/mL LPS). Data shown represents mean ± SEM of n=4 biological replicates. Statistical analysis was performed by unpaired t-test in comparison to untreated control, significance level: 10 %, significance is indicated by asterisk.


We also assed the basal promoter activity and compared it to the CMV promoter activity (see figure 2). The IL-6 promoter shows a comparable basal activity with the CMV promoter. This behavior is surprising but can be possibly explained by differences in transfection efficiencies of both promoter constructs. However, further experiments are needed to clarify.


Figure 2: Relative basal activity of all tested promoters in HeLa cells 48 hours (A) and 72 hours (B) after transfection. Data was normalized to CMV promoter which served as reference. Data shown represents mean ± SEM of n=4 biological replicates.


Summary

In summing up, the interleukin-6 promoter exhibits a significant LPS-sensitivity and a low basal activity making it ideal for use as a LPS (inflammatory toxin-) sensor.


References

Beetz, A., Peter, R. U., Oppel, T., Kaffenberger, W., Rupec, R. A., Meyer, M., van Beuningen, D., Kind, P., & Messer, G. (2000). NF-kappaB and AP-1 are responsible for inducibility of the IL-6 promoter by ionizing radiation in HeLa cells. International journal of radiation biology, 76(11), 1443–1453.

Heinrich, P. C., Behrmann, I., Haan, S., Hermanns, H. M., Müller-Newen, G., & Schaper, F. (2003). Principles of interleukin (IL)-6-type cytokine signalling and its regulation. The Biochemical journal, 374(Pt 1), 1–20.

Kawai, T., & Akira, S. (2007). Signaling to NF-kappaB by Toll-like receptors. Trends in molecular medicine, 13(11), 460–469.

Liu, X., Yin, S., Chen, Y., Wu, Y., Zheng, W., Dong, H., Bai, Y., Qin, Y., Li, J., Feng, S., & Zhao, P. (2018). LPS‑induced proinflammatory cytokine expression in human airway epithelial cells and macrophages via NF‑κB, STAT3 or AP‑1 activation. Molecular medicine reports, 17(4), 5484–5491.

Xiao, W., Hodge, D. R., Wang, L., Yang, X., Zhang, X., & Farrar, W. L. (2004). NF-kappaB activates IL-6 expression through cooperation with c-Jun and IL6-AP1 site, but is independent of its IL6-NFkappaB regulatory site in autocrine human multiple myeloma cells. Cancer biology & therapy, 3(10), 1007–1017.