Part:BBa_K3338002

Synthetic promoter_2 with NF-κB and AP1 binding sites

Usage and Biology

The presented synthetic promoter is able to recognize LPS in the surrounding of the cell via the Toll-like receptor, NF-κB/AP1 pathway leading to strong induction of gene expression. Due to its relatively low basal activity and strong induction following LPS supplementation it is perfectly suitable for the application as a LPS-sensor. It was designed as an improvement of the CMV promoter making it LPS-sensing. This improvment is also documented on the wiki page of the original part BBa_I712004. The novel synthetic promoter is based on a minimal CMV-promoter just containing the TATA-box and the Initiator-Sequence of the original CMV-promoter. Upstream of the minimal Promoter three repetitions of randomly generated AP1-NF-κB binding sites are localized.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1
  Illegal NotI site found at 58
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Improvement from the iGEM-team Hannover 2020

Overview

As an improvement of the natural CMV-promoter that shows no activation following LPS-stimulation, we designed a promoter based on the CMV-promoter that displays high activation after LPS-stimulation. Shortly, we replaced the CMV-enhancer and large parts of the promoter by NF-κB and AP1 binding sites just leaving the TATA-box and the Initiator-Sequence of the original CMV-promoter. The improved part and a full description of the improvement can also be found here (BBa_K3338002). Using the same approach, we have developed a promoter that shows 25-fold basal expression of the CMV enhancer/promoter named Synthetic promoter 1 (BBa_K3338007).

Motivation

One of the central aspects of our inflammatory toxin sensor is the LPS-sensitivity of its promoter. Because of that, one of our goals was to design a LPS-sensitive promoter showing high induction following LPS-treatment. In a previous study it was shown that the human CMV immediate‐early (CMV-IE) gene enhancer/promoter shows NF-κB and c-Jun dependent regulation in response to LPS and bacterial CpG‐oligodeoxynucleotides (Lee et al. 2004). The experiments were performed in the macrophage cell line RAW 264.7 and showed that a maximum of CMV induction was reached 6\,h after LPS-supplementation followed by a decrease in promoter activity (Lee et al. 2004). This observation was not reproducible in HeLa-cells using a CMV-hGLuc construct (BBa_K3338017) in the mammalian expression vector pEGFP-C2 (see figure 1). T--Hannover--results_promoter_CMV: Figure 1: Relative activity of CMV 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. To achieve high induction of the CMV-promoter following LPS-treatment of the cells, we had to modify the promoter/enhancer-regions of the CMV-promoter.

Part Design

LPS-sensitivity of the CMV-promoter is mediated through NF-κB and AP1 transcription factor binding sites (Lee et al. 2004). In order to abolish effects of other transcription factors on promoter activity, the CMV-enhancer and large parts of the promoter were removed just leaving a minimal CMV promoter with the TATA-box and the initiator-sequence which are essential for promoter activity. To make the promoter sensing, we added AP1 and NF-κB transcription factor binding sites right in front of the CMV_min promoter. These sequences were generated using a MATLAB tool which creates random AP1 and NF-κB sites based on the consensus sequence “GGGRNYYYCC” for NF-κB and “TGAXTCA” for AP1, where Y is C or T, R is A or G, X is C or G and N is any base (Chen et al. 1998, Alam and Den 1992). Two variants were built (see figure 2). One exhibiting three NF-κB sites followed by three AP-1 sites (Synthetic promoter_1, BBa_K3338007) and another one consisting of three NF-κB/AP-1-sites (Synthetic promoter_2, BBa_K3338002).

T--Hannover--results_synthpromoters: Figure 2: Schematic representation of the generation of the NF-κB-AP1-minCMV promoters. Combination of both transcription factor binding site sequences (3 times each) and the minimal CMV promoter (A) yielded the two synthetic promoter sequences used for our project (B).

Results

In order to test the synthetic promoters described above, their activity following LPS-stimulation was ascertained using hGLuc as a reporter gene. Therefore SynthP_1-hGLuc (BBa_K3338021) and SynthP_2-hGLuc (BBa_K3338022) were cloned into pEGFP-C2 using AseI/HindIII to linearize the vector (CMV-enhancer/promoter and EGFP were removed). The constructs were transfected in HeLa cells. The cells were treated with LPS for 3 h and hGLuc expression was assessed 24 h and 48 h after LPS-treatment. Figure 3 and figure 4 show quantitative data of the experiments. T--Hannover--results_promoter_minCMV_1: Figure 3: Relative activity of NF-κB-AP1-minCMV1 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.

T--Hannover--results_promoter_minCMV_2: Figure 4: Relative activity of NF-κB-AP1-minCMV2 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.

For both synthetic promoters a significant upregulation of the promoter activity was observed 24 h and 48 h after LPS-treatment of the cells for 3 h. Whereas the activity of the synthetic promoter_1 maximally increases about 40 % over the activity of the untreated cells, the activity of the synthetic promoter_2 rises up to approximately 100 % over the basal activity. For both promoters, the highest values were observed for cells treated with 2 μg/mL LPS. Furthermore, the basal activities of both synthetic promoters were compared (see figure 5) using the basal activity of the CMV promoter as a standard (see figure 5).

T--Hannover--results_promoter_basal: Figure 5: 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.

The results indicate that both synthetic promoters exhibit a higher basal activity than the original CMV-promoter. This could be due to higher transfection efficiencies as compared to the original CMV-hGLuc construct because of the lesser plasmid size. However, further experiments are needed to clarify. The naturally occurring LPS-sensitive human IL-6 promoter shows a basal activity comparable with the CMV-promoter. For the usage as a LPS-sensor the promoter ideally exhibits a low basal activity like the IL-6 promoter. But even more important is its LPS sensitivity. Figure 6 indicates that the gain of IL-6 activity is significant but much smaller compared to the synthetic promoters.

T--Hannover--results_promoter_IL6: Figure 6: 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.

Conclusions

Here we describe BBa_K3338002 as an improved CMV-promoter that is capable of detecting LPS in the surrounding of the cell via the TLR4-NF-κB/c-Jun pathway leading to enhanced gene expression. It is much more sensitive than the naturally occurring LPS-sensitive interleukin-6 promoter but shows a higher basal activity than the IL-6 and the CMV-promoter. Regarding the basal activity and LPS-sensitivity the Synthetic promoter_2 has tremendous advantages over Synthetic promoter_1 making it the better LPS-sensor. But it is of note that the basal activity of the Synthetic promoter_1 is up to approximately 25-times higher than of the natural CMV-promoter. This tremendous enhancement of promoter activity makes it applicable for protein expression in mammalian expression systems when maximum protein quantities are required.

References

Alam, J., & Den, Z. (1992). Distal AP-1 binding sites mediate basal level enhancement and TPA induction of the mouse heme oxygenase-1 gene. The Journal of biological chemistry, 267(30), 21894–21900.

Chen, F. E., Huang, D. B., Chen, Y. Q., & Ghosh, G. (1998). Crystal structure of p50/p65 heterodimer of transcription factor NF-kappaB bound to DNA. Nature, 391(6665), 410–413.

Lee, Y., Sohn, W. J., Kim, D. S., & Kwon, H. J. (2004). NF-kappaB- and c-Jun-dependent regulation of human cytomegalovirus immediate-early gene enhancer/promoter in response to lipopolysaccharide and bacterial CpG-oligodeoxynucleotides in macrophage cell line RAW 264.7. European journal of biochemistry, 271(6), 1094–1105.