Difference between revisions of "Part:BBa K5317021"
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===Usage and Biology===
 
===Usage and Biology===
  
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ATF2 belongs to the ATF/CREB family and regulates genes involved in cell growth, stress responses and apoptosis. With the addition of the ATF2 gene, this plasmid enables the study of transcriptional regulation of ATF2 (Kirsch ''et al.'', 2020) and its phosphorylation by PknB, making it important for research into signaling pathways related to cell stress and survival. It also could play a role in modulating bacterial virulence by altering key regulatory proteins, affecting their role in infection or antibiotic resistance. (Zhang ''et al.'', 2020) while mRuby2 provides a fluorescent marker for visualization. In our cell-based & #946;-lactam ring-containing antibiotics sensor, ATF2 serves as a translator of changes in PknB activity at the level of gene regulation, in particular the activity of the ATF2-3xCre2xAP1 promoter.
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ATF2 belongs to the ATF/CREB family (Kirsch ''et al.'', 2020) and its phosphorylation by PknB, making it important for research into signaling pathways related to cell stress and survival, while mRuby2 provides a fluorescent marker for visualisation. In our cell-based & #946;-lactam ring-containing antibiotics sensor, ATF2 serves as a translator of changes in PknB activity at the level of gene regulation, in particular the activity of the ATF2-3xCre2xAP1 promoter.
  
 
=Cloning=
 
=Cloning=
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===Theoretical Part Design===
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We placed the mRuby2 fluorescent marker (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317001 K5317001]</span>) downstream behind ATF2 (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317015 K5317015]</span>). This gene was codon optimised for human cell lines. This part was amplified by using the primers in table 1.
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<html>
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  <head>
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      <title>HTML Table Caption</title>
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  </head>
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<body>
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<caption>Table1: Primers used to extract the ATF2 gene sequence.</caption>
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<table style="width:70%">
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  <tr>
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    <th>Primer name</th>
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    <th>Sequence</th>
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  </tr>
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  <tr>
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    <td>ATF2_fw_1</td>
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    <td>TGAACCGTCAGATCCGatgaaattcaagttacatgtgaattctgccag</td>
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  </tr>
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  <tr>
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    <td>ATF2_rv_2</td>
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    <td>ggatccccacttcctgagggctgtgac</td>
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</tr>
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  <tr>
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    <td>ATF2_fw_3</td>
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    <td>caggaagtggggatccaccggtcg</td>
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  </tr>
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  <tr>
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    <td>ATF2_rv_4</td>
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    <td>TCAGTTATCTAGATCCGGTGcttgtacagctcgtccatccc</td>
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  </tr>
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</table>
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</body>
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===Sequence and features===
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<partinfo>BBa_K5317021 </partinfo>
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===Cloning===
 
Furthermore, the CMV promoter ensures robust  
 
Furthermore, the CMV promoter ensures robust  
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expression in mammalian systems (Radhakrishnan ''et al.'', 2008) that can be easily detected and analysed. This composite part was engineered with NEBBuilder® HIFI assembly method. We linearized eGFP-C2 with BamHI and AseI inserting ATF2 and mRuby.  
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expression in mammalian systems (Radhakrishnan ''et al.'', 2008) that can be easily detected and analysed. This composite part was engineered with NEBBuilder® HIFI assembly method. We linearized eGFP-C2 with BamHI and AseI inserting a linked ATF2 and mRuby seamlessly.  
  
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=Characterisation=
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K5317021 SequenceAndFeatures</partinfo>
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===References===
 
===References===
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Radhakrishnan, P., Basma, H., Klinkebiel, D., Christman, J., & Cheng, P.-W. (2008). Cell type-specific activation of the cytomegalovirus promoter by dimethylsulfoxide and 5-Aza-2’-deoxycytidine. ''The International Journal of Biochemistry & Cell Biology'', 40(9), 1944–1955. https://doi.org/10.1016/j.biocel.2008.02.014
 
Radhakrishnan, P., Basma, H., Klinkebiel, D., Christman, J., & Cheng, P.-W. (2008). Cell type-specific activation of the cytomegalovirus promoter by dimethylsulfoxide and 5-Aza-2’-deoxycytidine. ''The International Journal of Biochemistry & Cell Biology'', 40(9), 1944–1955. https://doi.org/10.1016/j.biocel.2008.02.014
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Zhang, C., Zhang, R., Dai, X., Cao, X., Wang, K., Huang, X., & Ren, Q. (2020). Activating transcription factor 2 (ATF2) negatively regulates the expression of antimicrobial peptide genes through tumor necrosis factor (TNF) in Macrobrachium nipponense. ''Fish & Shellfish Immunology'', 107, 26–35. https://doi.org/10.1016/j.fsi.2020.09.043
 
  
  

Revision as of 16:51, 27 September 2024


CMV-ATF2-mRuby2

Usage and Biology

ATF2 belongs to the ATF/CREB family (Kirsch et al., 2020) and its phosphorylation by PknB, making it important for research into signaling pathways related to cell stress and survival, while mRuby2 provides a fluorescent marker for visualisation. In our cell-based & #946;-lactam ring-containing antibiotics sensor, ATF2 serves as a translator of changes in PknB activity at the level of gene regulation, in particular the activity of the ATF2-3xCre2xAP1 promoter.

Cloning

Theoretical Part Design

We placed the mRuby2 fluorescent marker (K5317001) downstream behind ATF2 (K5317015). This gene was codon optimised for human cell lines. This part was amplified by using the primers in table 1.

HTML Table Caption Table1: Primers used to extract the ATF2 gene sequence.

Primer name Sequence
ATF2_fw_1 TGAACCGTCAGATCCGatgaaattcaagttacatgtgaattctgccag
ATF2_rv_2 ggatccccacttcctgagggctgtgac
ATF2_fw_3 caggaagtggggatccaccggtcg
ATF2_rv_4 TCAGTTATCTAGATCCGGTGcttgtacagctcgtccatccc
===Sequence and features=== BBa_K5317021 ===Cloning=== Furthermore, the CMV promoter ensures robust expression in mammalian systems (Radhakrishnan ''et al.'', 2008) that can be easily detected and analysed. This composite part was engineered with NEBBuilder® HIFI assembly method. We linearized eGFP-C2 with BamHI and AseI inserting a linked ATF2 and mRuby seamlessly. =Characterisation= ===References=== Kirsch, K., Zeke, A., Tőke, O., Sok, P., Sethi, A., Sebő, A., Kumar, G. S., Egri, P., Póti, Á. L., Gooley, P., Peti, W., Bento, I., Alexa, A., & Reményi, A. (2020). Co-regulation of the transcription controlling ATF2 phosphoswitch by JNK and p38. ''Nature Communications'', 11(1), 5769. https://doi.org/10.1038/s41467-020-19582-3 Radhakrishnan, P., Basma, H., Klinkebiel, D., Christman, J., & Cheng, P.-W. (2008). Cell type-specific activation of the cytomegalovirus promoter by dimethylsulfoxide and 5-Aza-2’-deoxycytidine. ''The International Journal of Biochemistry & Cell Biology'', 40(9), 1944–1955. https://doi.org/10.1016/j.biocel.2008.02.014