Latest revision as of 10:36, 2 October 2024

3xCre3xAP1-miniCMV-miRFP670

Usage and Biology

When ß-lactams bind to the PASTA domain of PknB, its kinase domain phosphorylates ATF2, which then binds to our promoter. The promoter was identified by (Miller et al., 2010) as a cyclic AMP responsive element (Cre)-sequence. ATF2-binding site is a consensus: 5-GTGACGT[AC][AG]-3) cAMP response element (CRE) (Hai et al., 1989). Based on observations made by Miller and colleagues (2010) showing similar kinase mechanisms between the prokaryotic PknB and eukaryotic MAPK towards ATF2, we generated a synthetic ATF2-responsive promoter construct with three Cre and three AP1 binding sites as well as a miniCMV promoter sequence. ATF2 was identified as the best PknB interaction partner. As with all our constructs, our promoter is followed by a fluorescent marker gene miRFP670 to detect specific activation.

Cloning

Theoretical Part Design

We generated a promoter sequence containing three CRE-binding motifs as well as three AP1-binding sites followed by the minimal CMV (miniCMV) promoter. The miniCMV promoter contains the TATA-box and the Initiator-Sequence of the original CMV-promoter, ensuring a successful transcription, but in parallel allowing for specific expression dependent on upstream laying individually chosen binding sites. The sequence was synthesized with approx. 20 bp-long overhangs to allow for correct orientation when integrated upstream of a reporter gene in a plasmid backbone. We chose to place the miRFP670 fluorescent marker (K5317002) downstream of the synthetic promoter, since miRFP with its excitation maxima at 642 nm and emission maxima at 670 nm is well suited to be imaged next to EGFP and mRuby2, both implemented in our sensor system.

Sequence and Features

Cloning

This plasmid was engineered with NEBBuilder HIFI assembly method. First, the backbone eGFP-C2neo was linearized with AseI and BamHI, creating matching overhangs to the synthesized 3xCre3xAP1-miniCMV sequence and the miRFP670 gene in the backbone ensured seamless cloning with predetermined order. We have designed this sequence containing triplicates of the Cre binding site as well as the AP1 binding site to increase the signal intensity. To ensure an optimal gene expression of miRFP670 we cloned a miniCMV promoter behind the recognition sequence of ATF2. The promoter insert was synthesized with the correct overhangs already. The miRFP670 reporter gene was amplified by PCR using the primers in table 1.

Table 1: Primers used to amplify miRFP670 with matching 3' and 5' overhangs.

Figure 1: Assembled vector map with 3xCre3xAP1-miniCMV-miRFP670 integrated into the pEGFP-C2 backbone.

Characterisation

Transfection experiments in mammalian HEK293T cells assessed the promoter functionality, sensitivity, and specificity. The fully assembled cell-based beta-lactam-sensor requires the successful triple-transduction with the detecting unit, CMV-EGFP-PknB, the signal transmitting unit, CMV-ATF2-mRuby2, and the receiving and signal translating into a fluorescence signal unit, the promoter 3xCre3xAP1-miniCMV-miRFP670. However, before all three are introduced into the cell and tested with antibiotic stimulation, it is necessary to test them alone and control for any sensor-unrelated responses of the promoter.

Single-transfection experiments

The composite part carrying plasmid was introduced via transfection to establish a baseline of endogenous promoter activity before performing co-transfection experiments with the CMV-ATF2-mRuby2 (composite part K5317016) and CMV-EGFP-PknB carrying plasmid (composite part K5317013) for ampicillin stimulation. The EGFP, mRuby2, and miRFP650 fluorescence signals were analyzed for presence and localization by microscopy.

Figure 2: Representative microscopic images of HEK293T cells expressing 3xCre3xAP1-miniCMV-miRFP670. Fluorescence channels for EGFP, mRuby2, and miRFP670 are shown. The basal activity of the promoter is presented in the upper row. The promoter activity after induction with 100 µg/mL ampicillin for four hours is demonstrated in the lower row. Scale bar = 100 µm.

The single-transfection experiment with the promoter and with or without beta-lactams demonstrated no unspecific signals from the miRFP670 in the green or lower red channels. The specific miRFP670 signal did not increase after incubation with ampicillin as expected since the signal detecting and transmitting units are not present in the cell. Rather fewer cells demonstrated a miRFP670 signal with ampicillin incubation. But it is important that this could result from different transfection efficiencies in the representative wells.

Triple-transfection experiments

To test the functionality of the cell-based beta-lactam-sensor, HEK293T cells were triple-transfected with all necessary parts for signal detection (EGFP-PknB), signal transmission (ATF2-mRuby2) and signal emittance (3xCre3xAP1-miniCMV-miRFP670) and the fluorescence signals analyzed via microscopy and later on FACS analysis.

Figure 3: Representative microscopy image of HEK293T cells expressing EGFP-PknB, ATF2-mRuby2 and 3xCre3xAP1-miniCMV-miRFP670. Shown are the fluorescence channels for EGFP, mRuby2, and miRFP670 (first three images from the left) and an overlay of the three channels (right). In a) is shown the basal activity of the promoter. In b) is shown the promoter activity after induction with 100 µg/mL ampicillin after four hours of incubation.

The representative images in figure 3 show nicely the correct localizations of all three sensor components with EGFP-PknB being mostly localized in the membrane regions, the mRuby2-ATF2 signal being emitted from the nucleus, and the miRFP670 from the nucleocytoplasm. The detectable miRFFP670 signal even without the presence of ampicillin could be explained by possible binding of ATF2 to the 3xCre3xAP1-sites after its activation via other mammalian mechanisms, since ATF2 is a mammalian transcription factor and possibly endogenously expressed and activated or other AP proteins are present, binding to our promoter. Nevertheless, the representative images of the miRFP670 channel indicate an increase in fluorescence intensity after ampicillin incubation suggesting a functional PASTA domain activity followed by ATF2 phosphorylation leading to miRFP670 expression.

FACS Analysis

FACS analysis enables the quantification of the miRFP670-positive cells. To exclude single- and double-transfected cells from the evaluation, the cells were pregated regarding their EGFP- and mRuby2-positivity, counting only cells that carry the CMV-EGFP-PknB-C2, CMV-ATF2-mRuby2-C2 and promoter plasmid. The cells were incubated with varying, increasing concentrations of ampicillin to stimulate the miRFP670 expression via our sensor cascade.

Figure 4: Quantitive validation of reporter activity by flow cytometry analysis. The percentage of cells expressing the fluorophore miRFP670 under the control of the tested 3xCre3xAP1-miniCMV promoter is displayed as a function of various concentrations of ampicillin. n=1.

The quantification of the miRFP670-posive cells dependent on the beta-lactam concentration present in the medium for four hours shows a high basal activity of the 3xCre3xAP1-miniCMV promoter (approx. 70 % miRFP670-positive cells), as mentioned, possibly caused by endogenously active ATF2 or other transcription factors interacting with Cre and/or AP1 proteins-binding sites, activating our promoter independent of the presence of ampicillin. Nevertheless, the percentage of miRFP670-expressing cells increased slightly with ampicillin supplementation of the culture media. The bars indicate that the sensitivity of the sensor-cassette, depicting already approx. 79 % miRFP670-positive cells under stimulation with 2.5 µg/mL ampicillin and no further increase with rising ampicillin concentrations. In conclusion, the evaluation of the sensor is made more difficult by a high baseline, but it is also characterized by a high sensitivity to beta-lactams. However, it must be taken into account that the experiment could only be carried out once due to time constraints.

References

Hai, T. W., Liu, F., Coukos, W. J., & Green, M. R. (1989). Transcription factor ATF cDNA clones: An extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers. Genes & Development, 3(12b), 2083–2090. https://doi.org/10.1101/gad.3.12b.2083

Miller, M., Donat, S., Rakette, S., Stehle, T., Kouwen, T. R. H. M., Diks, S. H., Dreisbach, A., Reilman, E., Gronau, K., Becher, D., Peppelenbosch, M. P., Van Dijl, J. M., & Ohlsen, K. (2010). Staphylococcal PknB as the First Prokaryotic Representative of the Proline-Directed Kinases. PLoS ONE, 5(2), e9057. https://doi.org/10.1371/journal.pone.0009057