Composite

Part:BBa_K5317018

Designed by: Lisa Marie de Sousa Miranda   Group: iGEM24_Hannover   (2024-09-15)
Revision as of 14:00, 29 September 2024 by Vanessa09 (Talk | contribs) (→‎Co-transfection experiments with ATF2)


CMV-EGFP-PknB

Usage and Biology

PknB is a eukaryote-like serine/threonine kinase in Staphylococcus aureus that plays an important role in the bacterial response to antibiotics, particularly beta-lactams, via its PASTA domain (Stehle et al.,2012). PknB is a membrane-localized protein consisting of an N-terminal cytosolic kinase domain, a central transmembrane segment and three C-terminal extracellular PASTA domains. The PASTA (penicillin-binding protein and serine/threonine kinase-associated) domain plays a critical role in the recognition and binding of beta-lactam antibiotics (Stehle et al.,2012). Upon binding these compounds, the PASTA domain initiates a signaling cascade by inducing autophosphorylation of the N-terminal kinase domain. This activation leads to the initiation of downstream signaling pathways (Cheung et al.,2010). In S. aureus, this mechanism is critical for early detection of antibiotics and helps the bacteria adapt to antibiotic stress (Sauer et al.,2018).

The composite part includes the upstream positioned reporter gene EGFP (K3338006) to charactarize the PknB regarding it's cellular localization pre and post antibiotics stimulation.

Cloning

Theoretical Part Design

Placing the PknB kinase upstream of the reporter gene EGFP allows the visualization of localisation of PknB.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 2596
    Illegal SpeI site found at 3259
    Illegal PstI site found at 2021
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 3259
    Illegal PstI site found at 2021
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2271
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 2596
    Illegal SpeI site found at 3259
    Illegal PstI site found at 2021
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 2596
    Illegal SpeI site found at 3259
    Illegal PstI site found at 2021
  • 1000
    COMPATIBLE WITH RFC[1000]

Cloning

PknB was synthesized and inserted by NEB HiFi Assembly into the pEGFP-C2 backbone plasmid (K3338020) after its restriction enzyme digestion with SaIHI and BamHI, generating the PknB-eGFP cassette.

HTML Table Caption Table1: Primers used to create matching overhangs of pknB amplicon to digested pEGFP-C2 backbone

Primer name Sequence
PknB_fw_1 AGCTTCGAATTCTGCAGAatgataggtaaaataataaatgaacgatataaaattgtagataagcttgg
PknB_rev_2 TCAGTTATCTAGATCCGGTGttatacatcatcatagctgacttctttttcagctacag

Figure 1: Assembled vector map with pknB-EGFP integrated into the pEGFP-C2 backbone.

Characterization

Transfection experiments in mammalian HEK293T cells assessed functionality of our PknB kinase localisation and sensitivity. The composite part carrying plasmid was introduced via transfection to establish cell localisation of pknB before performing co-transfection experiments with the CMV-ATF2-mRuby2 (K5317016) and ATF2-3xCre3xAP1-Promoter_miniCMV_miRFP670 K5317017) varying ampicillin concentration for stimulation. The EGFP fluorescence signal was analyzed for localization by microscopy and intensity by FACS analysis.

Single-transfection experiments

Figure 2: Single-transfected HEK293T cells with the PknB-EGFP-C2 plasmid depicted no EGFP-signal under unstimulated conditions. Scale bar = 20 µm.

As shown in figure 2, EGFP-PknB is correctly expressed in HEK293T cells. As intended, is the membranous installation of the codon-optimized prokaryotic membrane protein in the eukaryotic cell membrane.

Co-transfection experiments with ATF2

Figure 3: Representative microscopy image of HEK cells expressing EGFP-PknB and ATF2-mRuby2. Shown are brightfield (left), fluorescence channels for eGFP and mRuby2 (both images in the center) and an overlay of the three channels (right).

The co-transfection of the functional EGFP-PknB and ATF2-mRuby2 is shown in figure 3. The expression of both parts was detectable, which are also located in one cell.

Stimulation with Ampicillin

Figure 4: The montage double-transfected pknB and ATT2-Cre3x-API3x with and without ampicillin stimulation.

The co-transfection shows an image expressing HEK cells of EGFP with ATF2-Cre3x-API3x promotor. Shown are brightfield (left), fluorescence channels for eGFP and miRFP650 and an overlay of the three channels with and without coloured signals (right).

References

Pensinger, D. A., Schaenzer, A. J., & Sauer, J. D. (2018). Do Shoot the Messenger: PASTA Kinases as Virulence Determinants and Antibiotic Targets. Trends in microbiology, 26(1), 56–69. https://doi.org/10.1016/j.tim.2017.06.010

Rakette S, Donat S, Ohlsen K, Stehle T (2012) Structural Analysis of Staphylococcus aureus Serine/Threonine Kinase PknB. PLOS ONE 7(6): e39136. https://doi.org/10.1371/journal.pone.0039136

Tamber, S., Schwartzman, J., & Cheung, A. L. (2010). Role of PknB kinase in antibiotic resistance and virulence in community-acquired methicillin-resistant Staphylococcus aureus strain USA300. Infection and immunity, 78(8), 3637–3646. https://doi.org/10.1128/IAI.00296-10

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