Difference between revisions of "Part:BBa K5317020"
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===Usage and Biology=== | ===Usage and Biology=== | ||
− | + | Integrated into the CMV-GraR-mRuby2 cassette are two key genes next to the constitutively active CMV promoter: mRuby2, a red fluorescent protein for live-cell imaging, and GraR, a regulator that might bind to the PknB-kinase upon detecting beta-lactams and gets activated by phosphorylation. GraR is known for its role in β-lactam resistance by upregulating cell wall biosynthesis genes, altering cell wall composition, and increasing expression of ABC-transporter (El-Halfawy ''et al.'', 2020),(Yang ''et al.'', 2012),(Meehl ''et al.'', 2007). The GraSR system was found to control genes involved in stress response, cell wall metabolism, and virulence pathways, in addition to playing an important role in CAMP resistance (Falord ''et al.'', 2011). | |
+ | |||
+ | Our aim was its utilization as a signal transmitter in our cell-based antibiotic sensor. When phosphorylated by the detection unit PknB (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317013 K5317013]</span>), GraR translocates into the nucleus and binds to specific DNA sequences in our engineered 3xCre3xAP1-miniCMV promoter (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317017 K5317017]</span>), leading to the expression of the downstream located miRFP670 (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317002 K5317002]</span>). | ||
=Cloning= | =Cloning= | ||
===Theoretical Part Design=== | ===Theoretical Part Design=== | ||
− | + | The GraR sequence (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317015 K5317015]</span>) was codon-optimized for mammalian expression systems and synthesized before fusing the mRuby2 fluorescent marker (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317001 K5317001]</span>) C-terminally to visualize localisation of GraR when stimulated with ß-lactam antibiotics. To ensure a constitutive expression of the transcription factor, the CMV of the pEGFP-C2 backbone was utilized. | |
− | + | ||
===Sequence and features=== | ===Sequence and features=== | ||
Line 18: | Line 19: | ||
===Cloning=== | ===Cloning=== | ||
− | This | + | This part was engineered with NEBBuilder® HIFI assembly method. First, the backbone was linearized with NheI and BamHI, creating matching approx. 20 bp-long overhangs between both inserts and the backbone ensuring the correct order of CMV-GraR-mRuby2. In mammalian systems, we used this plasmid to study the potential interactions between GraR and PknB. The inserts were amplified by using the primers in table 1. |
<html> | <html> | ||
Line 34: | Line 35: | ||
<body> | <body> | ||
− | <caption>Table1: Primers used to extract the | + | <caption>Table1: Primers used to extract the GraR gene sequence.</caption> |
<table style="width:70%"> | <table style="width:70%"> | ||
Line 48: | Line 49: | ||
<tr> | <tr> | ||
− | <td> | + | <td>GraR_fw</td> |
<td>TGAACCGTCAGATCCGatgcaaatactactagtagaagatgacaatactttgt</td> | <td>TGAACCGTCAGATCCGatgcaaatactactagtagaagatgacaatactttgt</td> | ||
Line 56: | Line 57: | ||
<tr> | <tr> | ||
− | <td> | + | <td>GraR_rev</td> |
<td>tggatccccttcatgagccatatatccttttcctacttttgt</td> | <td>tggatccccttcatgagccatatatccttttcctacttttgt</td> | ||
Line 63: | Line 64: | ||
<tr> | <tr> | ||
− | <td> | + | <td>mRuby2_fw</td> |
<td>tggatccccttcatgagccatatatccttttcctacttttgt</td> | <td>tggatccccttcatgagccatatatccttttcctacttttgt</td> | ||
Line 71: | Line 72: | ||
<tr> | <tr> | ||
− | <td> | + | <td>mRuby2_rev</td> |
<td>TCAGTTATCTAGATCCGGTGttacttgtacagctcgtccatcccacc</td> | <td>TCAGTTATCTAGATCCGGTGttacttgtacagctcgtccatcccacc</td> | ||
Line 94: | Line 95: | ||
</html> | </html> | ||
− | Figure 1: Assembled vector map with | + | Figure 1: Assembled vector map with GraR-mRuby2 integrated into the pEGFP-C2 backbone. |
=Characterisation= | =Characterisation= | ||
+ | Transfection experiments were conducted of CMV-GraR-mRuby2 in mammalian HEK293T cells to show successful expression and localization of GraR under unstimulated conditions. | ||
+ | |||
+ | ===Single-transfection experiments=== | ||
+ | |||
+ | <html> | ||
+ | <center> | ||
+ | <img src="https://static.igem.wiki/teams/5317/grar-bba-k5317020-2.png" style="width: 70%; height: 70%"> | ||
+ | </p> | ||
+ | </center> | ||
+ | </html> | ||
+ | |||
+ | Figure 2: Single-transfected HEK293T cells with the CMV-graR-mRuby2 plasmid depicted low mRuby2-signal under unstimulated conditions. Scale bar = 20 µm. | ||
+ | |||
+ | The representative images of figure 2 depict HEK293T cells after transfection with CMV-GraR-mRuby2. Even under unstimulated conditions, the HEK293T cells emitted low mRuby2 fluorescent signals, indicating a successful codon optimization and transfection. No further experiments were performed using this transcription factor since the intracellular assembly of all sensor parts was performed using the transcription factor ATF2 (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317016 K5317016]</span>) as the signal transmitter. | ||
=References= | =References= |
Latest revision as of 21:04, 1 October 2024
CMV-GraR-mRuby2
Usage and Biology
Integrated into the CMV-GraR-mRuby2 cassette are two key genes next to the constitutively active CMV promoter: mRuby2, a red fluorescent protein for live-cell imaging, and GraR, a regulator that might bind to the PknB-kinase upon detecting beta-lactams and gets activated by phosphorylation. GraR is known for its role in β-lactam resistance by upregulating cell wall biosynthesis genes, altering cell wall composition, and increasing expression of ABC-transporter (El-Halfawy et al., 2020),(Yang et al., 2012),(Meehl et al., 2007). The GraSR system was found to control genes involved in stress response, cell wall metabolism, and virulence pathways, in addition to playing an important role in CAMP resistance (Falord et al., 2011).
Our aim was its utilization as a signal transmitter in our cell-based antibiotic sensor. When phosphorylated by the detection unit PknB (K5317013), GraR translocates into the nucleus and binds to specific DNA sequences in our engineered 3xCre3xAP1-miniCMV promoter (K5317017), leading to the expression of the downstream located miRFP670 (K5317002).
Cloning
Theoretical Part Design
The GraR sequence (K5317015) was codon-optimized for mammalian expression systems and synthesized before fusing the mRuby2 fluorescent marker (K5317001) C-terminally to visualize localisation of GraR when stimulated with ß-lactam antibiotics. To ensure a constitutive expression of the transcription factor, the CMV of the pEGFP-C2 backbone was utilized.
Sequence and features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 889
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 889
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 889
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1342
Cloning
This part was engineered with NEBBuilder® HIFI assembly method. First, the backbone was linearized with NheI and BamHI, creating matching approx. 20 bp-long overhangs between both inserts and the backbone ensuring the correct order of CMV-GraR-mRuby2. In mammalian systems, we used this plasmid to study the potential interactions between GraR and PknB. The inserts were amplified by using the primers in table 1.
Primer name | Sequence |
---|---|
GraR_fw | TGAACCGTCAGATCCGatgcaaatactactagtagaagatgacaatactttgt |
GraR_rev | tggatccccttcatgagccatatatccttttcctacttttgt |
mRuby2_fw | tggatccccttcatgagccatatatccttttcctacttttgt |
mRuby2_rev | TCAGTTATCTAGATCCGGTGttacttgtacagctcgtccatcccacc |
Figure 1: Assembled vector map with GraR-mRuby2 integrated into the pEGFP-C2 backbone.
Characterisation
Transfection experiments were conducted of CMV-GraR-mRuby2 in mammalian HEK293T cells to show successful expression and localization of GraR under unstimulated conditions.
Single-transfection experiments
Figure 2: Single-transfected HEK293T cells with the CMV-graR-mRuby2 plasmid depicted low mRuby2-signal under unstimulated conditions. Scale bar = 20 µm.
The representative images of figure 2 depict HEK293T cells after transfection with CMV-GraR-mRuby2. Even under unstimulated conditions, the HEK293T cells emitted low mRuby2 fluorescent signals, indicating a successful codon optimization and transfection. No further experiments were performed using this transcription factor since the intracellular assembly of all sensor parts was performed using the transcription factor ATF2 (K5317016) as the signal transmitter.
References
El-Halfawy, O. M., Czarny, T. L., Flannagan, R. S., Day, J., Bozelli, J. C., Kuiack, R. C., Salim, A., Eckert, P., Epand, R. M., McGavin, M. J., Organ, M. G., Heinrichs, D. E., & Brown, E. D. (2020). Discovery of an antivirulence compound that reverses β-lactam resistance in MRSA. Nature Chemical Biology, 16(2), 143–149. https://doi.org/10.1038/s41589-019-0401-8
Falord, M., Mäder, U., Hiron, A., Débarbouillé, M., & Msadek, T. (2011). Investigation of the Staphylococcus aureus GraSR Regulon Reveals Novel Links to Virulence, Stress Response and Cell Wall Signal Transduction Pathways. PLoS ONE, 6(7), e21323. https://doi.org/10.1371/journal.pone.0021323
Meehl, M., Herbert, S., Götz, F., & Cheung, A. (2007). Interaction of the GraRS Two-Component System with the VraFG ABC Transporter To Support Vancomycin-Intermediate Resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy, 51(8), 2679–2689. https://doi.org/10.1128/AAC.00209-07
Yang, S.-J., Bayer, A. S., Mishra, N. N., Meehl, M., Ledala, N., Yeaman, M. R., Xiong, Y. Q., & Cheung, A. L. (2012). The Staphylococcus aureus Two-Component Regulatory System, GraRS, Senses and Confers Resistance to Selected Cationic Antimicrobial Peptides. Infection and Immunity, 80(1), 74–81. https://doi.org/10.1128/IAI.05669-11