Difference between revisions of "Part:BBa K5317018"
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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 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). We utilized the PknB protein as the beta-lactam detector that passes the signal by phosphorylating one of our three transcription factors ATF2 (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317016 K5317016]</span>), GraR (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317015 K5317015]</span>) or CcpA (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317014 K5317014]</span>). | + | 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 the early detection of antibiotics and helps the bacteria adapt to antibiotic stress (Sauer ''et al.'',2018). We utilized the PknB protein as the beta-lactam detector that passes the signal by phosphorylating one of our three transcription factors ATF2 (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317016 K5317016]</span>), GraR (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317015 K5317015]</span>) or CcpA (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317014 K5317014]</span>). |
| − | The composite part includes the upstream positioned constitutive active promoter CMV and the reporter gene EGFP (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K3338006 K3338006]</span>) to | + | The composite part includes the upstream positioned constitutive active promoter CMV and the reporter gene EGFP (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K3338006 K3338006]</span>) to characterize the PknB regarding its cellular localization pre and post antibiotics stimulation. |
=Cloning= | =Cloning= | ||
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===Theoretical Part Design=== | ===Theoretical Part Design=== | ||
| − | The CMV promoter was chosen to ensure a constitutive | + | The CMV promoter was chosen to ensure a constitutive expression of the PknB in HEK293T cells and placing the PknB kinase upstream of the reporter gene EGFP allows the visualization of localization of PknB. The PknB gene sequence itself was codon-optimized for expression in mammalian systems. |
===Sequence and Features=== | ===Sequence and Features=== | ||
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| − | The PknB insert was | + | The PknB insert was synthesized and the PknB sequence was amplified using the primers outlined in Table 1. The primers ensured that the 5' and 3' ends of the amplicons exhibited approximately 20 base pair (bp) overhangs, rendering them compatible with the backbone. The EGFP-C2 plasmid was linearized with BamHI and SalHI. The composite part-containing plasmid was assembled using the NEBBuilder® HIFI assembly kit, ensuring the correct positioning of the insert in the backbone through the use of overhangs. |
<html> | <html> | ||
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=Characterization= | =Characterization= | ||
| − | Transfection experiments in mammalian HEK293T cells assessed | + | Transfection experiments in mammalian HEK293T cells assessed the functionality of our PknB kinase localization and sensitivity. The composite part carrying plasmid was introduced via transfection to establish cellular localization of PknB before performing co-transfection experiments with the CMV-ATF2-mRuby2 (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317016 K5317016]</span>) and 3xCre3xAP1-miniCMV-miRFP670 promoter <span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317017 K5317017]</span>) under varying ampicillin concentration for stimulation. The EGFP fluorescence signal was analyzed for localization by microscopy and intensity by FACS analysis. |
===Single-transfection experiments=== | ===Single-transfection experiments=== | ||
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Figure 2: Single-transfected HEK293T cells with the PknB-EGFP-C2 plasmid depicted no EGFP-signal under unstimulated conditions. Scale bar = 20 µm. | 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, the EGFP-PknB depicts a membrane localized signal indicating a successful codon | + | As shown in figure 2, EGFP-PknB is correctly expressed in HEK293T cells. As intended, the EGFP-PknB depicts a membrane-localized signal indicating a successful codon optimization and correct implementation of a prokaryotic membrane protein into the eukaryotic cell membrane. With this, we were able to continue to find a functional detection protein, able to transfer the signal intracellularly. |
===Co-transfection experiments with ATF2 === | ===Co-transfection experiments with ATF2 === | ||
| − | To pass the detected signal intracellularly, a transcription factor is | + | To pass the detected signal intracellularly, a transcription factor is necessary that interacts with the kinase domain of PknB, and is activated by phosphorylation and transfers the signal on the level of expression regulation. Therefore, HEK293T cells were double-transfected with CMV-EGFP-PknB-C2 and CMV-ATF2-mRuby2 to analyze possible interactions by their fluorescence signals. Scale bar = 20 µM |
<html> | <html> | ||
| Line 107: | Line 107: | ||
</html> | </html> | ||
| − | Figure 3: Representative microscopy image of HEK293T 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). | + | Figure 3: Representative microscopy image of HEK293T 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, also located in one cell, indicating successful double-transfection. The EGFP signal, indicating the localization of PknB, was again membrane- | + | The co-transfection of the functional EGFP-PknB and ATF2-mRuby2 is shown in figure 3. The expression of both parts was detectable, also located in one cell, indicating successful double-transfection. The EGFP signal, indicating the localization of PknB, was again membrane-closely localized. ATF2-mRuby2 on the other side demonstrated a rather nuclear-cytoplasmic localization, both as expected. |
===Stimulation of Co-transfected PknB-EGFP and ATF2-mRuby2 HEK cells with ampicillin=== | ===Stimulation of Co-transfected PknB-EGFP and ATF2-mRuby2 HEK cells with ampicillin=== | ||
| Line 121: | Line 121: | ||
</html> | </html> | ||
| − | Figure 4: The montage shows representative images of double-transfected CMV-EGFP-PknB and CMV-ATF2-mRuby2 HEK293T cells with and without ampicillin stimulation. Shown are brightfield (left), fluorescence channels for eGFP and mRuby2 and an overlay of the three channels with and without | + | Figure 4: The montage shows representative images of double-transfected CMV-EGFP-PknB and CMV-ATF2-mRuby2 HEK293T cells with and without ampicillin stimulation. Shown are brightfield (left), fluorescence channels for eGFP and mRuby2, and an overlay of the three channels with and without colored signals (right). Scale bar = 100 µm. |
| − | The Co-transfection experiments showed that PknB and ATF2 do not inhibit each other's co-expression and that both are possibly interacting. Under ampicillin stimulating conditions, both signals increase slightly. | + | The Co-transfection experiments showed that PknB and ATF2 do not inhibit each other's co-expression and that both are possibly interacting. Under ampicillin-stimulating conditions, both signals increase slightly. |
| − | This | + | This pairing of kinase and transcription factor can be used for further experiments with the 3xCre3xAP1-miniCMV promoter to fully assemble the cell-based antibiotic sensor and test its responsiveness to beta-lactam exposure (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317022 K5317022]</span>). |
=References= | =References= | ||
Latest revision as of 11:33, 2 October 2024
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 the early detection of antibiotics and helps the bacteria adapt to antibiotic stress (Sauer et al.,2018). We utilized the PknB protein as the beta-lactam detector that passes the signal by phosphorylating one of our three transcription factors ATF2 (K5317016), GraR (K5317015) or CcpA (K5317014).
The composite part includes the upstream positioned constitutive active promoter CMV and the reporter gene EGFP (K3338006) to characterize the PknB regarding its cellular localization pre and post antibiotics stimulation.
Cloning
Theoretical Part Design
The CMV promoter was chosen to ensure a constitutive expression of the PknB in HEK293T cells and placing the PknB kinase upstream of the reporter gene EGFP allows the visualization of localization of PknB. The PknB gene sequence itself was codon-optimized for expression in mammalian systems.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 2596
Illegal SpeI site found at 3259
Illegal PstI site found at 2021 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 3259
Illegal PstI site found at 2021 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2271
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 2596
Illegal SpeI site found at 3259
Illegal PstI site found at 2021 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 2596
Illegal SpeI site found at 3259
Illegal PstI site found at 2021 - 1000COMPATIBLE WITH RFC[1000]
Cloning
The PknB insert was synthesized and the PknB sequence was amplified using the primers outlined in Table 1. The primers ensured that the 5' and 3' ends of the amplicons exhibited approximately 20 base pair (bp) overhangs, rendering them compatible with the backbone. The EGFP-C2 plasmid was linearized with BamHI and SalHI. The composite part-containing plasmid was assembled using the NEBBuilder® HIFI assembly kit, ensuring the correct positioning of the insert in the backbone through the use of overhangs.
| Primer name | Sequence |
|---|---|
| PknB_fw_1 | AGCTTCGAATTCTGCAGAatgataggtaaaataataaatgaacgatataaaattgtagataagcttgg |
| PknB_rev_2 | TCAGTTATCTAGATCCGGTGttatacatcatcatagctgacttctttttcagctacag |
Figure 1: Assembled vector map with CMV-EGFP-PknB integrated into the pEGFP-C2 backbone.
Characterization
Transfection experiments in mammalian HEK293T cells assessed the functionality of our PknB kinase localization and sensitivity. The composite part carrying plasmid was introduced via transfection to establish cellular localization of PknB before performing co-transfection experiments with the CMV-ATF2-mRuby2 (K5317016) and 3xCre3xAP1-miniCMV-miRFP670 promoter K5317017) under 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, the EGFP-PknB depicts a membrane-localized signal indicating a successful codon optimization and correct implementation of a prokaryotic membrane protein into the eukaryotic cell membrane. With this, we were able to continue to find a functional detection protein, able to transfer the signal intracellularly.
Co-transfection experiments with ATF2
To pass the detected signal intracellularly, a transcription factor is necessary that interacts with the kinase domain of PknB, and is activated by phosphorylation and transfers the signal on the level of expression regulation. Therefore, HEK293T cells were double-transfected with CMV-EGFP-PknB-C2 and CMV-ATF2-mRuby2 to analyze possible interactions by their fluorescence signals. Scale bar = 20 µM
Figure 3: Representative microscopy image of HEK293T 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, also located in one cell, indicating successful double-transfection. The EGFP signal, indicating the localization of PknB, was again membrane-closely localized. ATF2-mRuby2 on the other side demonstrated a rather nuclear-cytoplasmic localization, both as expected.
Stimulation of Co-transfected PknB-EGFP and ATF2-mRuby2 HEK cells with ampicillin
To show correct localization and interaction of PknB-EGFP and ATF2-mRuby2, both parts were transfected in HEK cells and incubated with ampicillin present in the culture media. We evaluated the fluorescence signal of these two proteins and their alteration of signal intensity compared to unstimulated, basal levels.
Figure 4: The montage shows representative images of double-transfected CMV-EGFP-PknB and CMV-ATF2-mRuby2 HEK293T cells with and without ampicillin stimulation. Shown are brightfield (left), fluorescence channels for eGFP and mRuby2, and an overlay of the three channels with and without colored signals (right). Scale bar = 100 µm.
The Co-transfection experiments showed that PknB and ATF2 do not inhibit each other's co-expression and that both are possibly interacting. Under ampicillin-stimulating conditions, both signals increase slightly.
This pairing of kinase and transcription factor can be used for further experiments with the 3xCre3xAP1-miniCMV promoter to fully assemble the cell-based antibiotic sensor and test its responsiveness to beta-lactam exposure (K5317022).
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