Difference between revisions of "Part:BBa K5317018"

(→‎Theoretical Part Design)
(→‎Cloning)
 
(10 intermediate revisions by 3 users not shown)
Line 6: Line 6:
  
 
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 charactarize the PknB regarding it's cellular localization pre and post antibiotics stimulation.
+
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=
Line 14: Line 14:
 
===Theoretical Part Design===
 
===Theoretical Part Design===
  
−
The CMV promoter was chosen to ensure a constitutive expresssion of the PknB in HEK293T cells and placing the PknB kinase upstream of the reporter gene EGFP allows the visualization of localisation of PknB. The PknB gene sequence itself was codon-optimized for expression in mammalian systems.
+
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===
Line 22: Line 22:
  
  
−
PknB was synthesized and inserted by NEB HiFi Assembly into the pEGFP-C2 backbone plasmid (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K3338020 K3338020]</span>) after its restriction enzyme digestion with SaIHI and BamHI, generating the PknB-eGFP cassette.  
+
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>  
Line 38: Line 38:
 
<body>  
 
<body>  
  
−
<caption>Table1: Primers used to create matching overhangs of pknB amplicon to digested pEGFP-C2 backbone</caption>  
+
<caption>Table1: Primers used to create matching overhangs of PknB amplicon to digested pEGFP-C2 backbone</caption>  
  
 
<table style="width:70%">  
 
<table style="width:70%">  
Line 79: Line 79:
 
</html>
 
</html>
  
−
Figure 1: Assembled vector map with PknB-EGFP integrated into the pEGFP-C2 backbone.
+
Figure 1: Assembled vector map with CMV-EGFP-PknB integrated into the pEGFP-C2 backbone.
  
 
=Characterization=
 
=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 (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317016 K5317016]</span>) and ATF2-3xCre3xAP1-Promoter_miniCMV_miRFP670  <span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317017 K5317017]</span>) varying ampicillin concentration for stimulation. The EGFP fluorescence signal was analyzed for localization by microscopy and intensity by FACS analysis.
+
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===
Line 95: Line 95:
 
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-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 transcription factor for signal transfer in the cell.
+
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 neccessary that interacts with the kinase domain of PknB, 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 analyse possible interactions by their fluorescence signal.  
+
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, which are also located in one cell. The EGFP signal, indicating the localization of PknB, was again membrane-closly localized. ATF2-mRuby2 on the other side demostrated a rather nuclear-cytoplasmic localization.
+
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.  
  
−
===Stimulation of Co-transfected PknB-EGFP and ATF2-mRuby2 HEK cells with Ampicillin===
 
−
To show correct localisation and interaction of PknB-eGFP and ATF2-mRruby2, both parts were transfected in HEK cells and  incubated under ampicillin stimulated conditions. We evaluated the fluorescence signal of these two proteins and their alteration of signal intensity compared to basal levels.
 
 
<html>
 
<html>
 
<center>
 
<center>
−
<img src="https://static.igem.wiki/teams/5317/atf2-stimulation-k5317018-2.png"style="width: 100%; height: 100%"></p>
+
<img src="https://static.igem.wiki/teams/5317/screenshot-2024-10-01-at-21-14-34.png"style="width: 100%; height: 100%"></p>
 
</center>
 
</center>
 
</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 colored signals (right). Scale bar = 100 µm.
  
−
Figure 4: The montage double-transfected CMV-PknB-eGFP and CMV-ATF2-mRuby2 in HEK cells with and without ampicillin stimulation in HEK cells with and without ampicillin stimulation.
 
  
−
The co-transfection (Figure 4) shows an image expressing HEK cells of CMV-PknB-eGFP and CMV-ATF2-mRuby2. Shown are brightfield (left), fluorescence channels for eGFP and mRuby2 and an overlay of the three channels with and without coloured signals (right). An increased signal of fluorescence can be measured of PknB and ATF2. However, this signal is controlled by the native human ATF2 promoter.
+
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 (<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


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

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 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 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