Revision as of 12:52, 29 September 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 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 placed the miRFP670 fluorescent marker (K5317002) downstream behind this synthetic promotor (K5317017).

Sequence and Features

Cloning

This plasmid was engineered with NEBBuilder HIFI assembly method. First the backbone eGFP-C2neo was lineraized with AseI and BamHI, matching ends of gene and backbone ensured seamless cloning. We have designed this sequence threefold and condon-optimised to increase the signal intensity. To ensure an optimal gene expression of miRFP670 we clonded behind the recognition sequenz of ATF1 a mini-CMV-Promotor to ensure better protein expression. This composite part was amplified by matching synthesised overhangs

Figure 1: Assembled vector map with ATF2-3xCre3xAP1-Promoter_miniCMV_miRFP670 integrated into the pEGFP-C2 backbone.

Characterisation

Transfection experiments in mammalian HEK293T cells assessed the promoter functionality, sensitivity and specifity. 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-EGFP (composite part K5317016) and CMV-PknB-mRuby2 carrying plasmid (composite part K5317013) under varying copper concentration for stimulation. The EGFP fluorescence signal was analyzed for localization by microscopy and intensity by FACS analysis.

Triple-transfection experiments

Figure 2: Representative microscopy image of HEK cells expressing EGFP-PknB, ATF2-mRuby2 and ATF2-3xCre3xAP1-Promoter_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.

FACS Analysis

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