Difference between revisions of "Part:BBa K1470005"
 
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<h2>Usage and Biology</h2>
 
<h2>Usage and Biology</h2>
 
<p> The engineered PDZ domain is a globuar protein domain of 80 - 90 amino acids wich has an enhanced interaction with other proteins. Normally PDZ domains bind to a short region at the C-terminus of other proteins but there are some proteins which recognize internal sequence motifs of target proteins through a single binding site on the domains [1][2]. There are more over 1000 known proteins containing this domain in eucaryotes and bacteria but just a few examples are shown in archea [3].<br>
 
<p> The engineered PDZ domain is a globuar protein domain of 80 - 90 amino acids wich has an enhanced interaction with other proteins. Normally PDZ domains bind to a short region at the C-terminus of other proteins but there are some proteins which recognize internal sequence motifs of target proteins through a single binding site on the domains [1][2]. There are more over 1000 known proteins containing this domain in eucaryotes and bacteria but just a few examples are shown in archea [3].<br>
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ePDZ is a crucial part of the blue light-inducible expression system. It is able to bind to the J-alpha-helix of the LOV2 domain. The principle of the light-induced expression is explained <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1150005">here</a> [4].  
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ePDZ is a crucial part of the blue light-inducible expression system. It is able to bind to the J-alpha-helix of the LOV2 domain. The principle of the light-induced expression is explained <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1150005">here</a> [4].
 
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<p>
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We obtained the ePDZ from Konrad Müller, AG Weber of the University of Freiburg. It was stardardized and biobricked by us.</p>
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<figure><img src="https://static.igem.org/mediawiki/2014/4/4f/Freiburg2014_results_blue_light_system_different_illuminations_seap.png" width="400px">
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<small><p><b>Figure 4: Efficiency of the blue light system using different time intervals of illumination.</b> To test the efficiency of the light system, SEAP expression after illumination was determined. The SEAP assay was performed 24 hours after light exposure. Cells were incubated with blue light for 1 hour, 2.5 hours and 5 hours.</p><a href="http://2014.igem.org/Team:Freiburg/Results/Light_system">More information</a></small>
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[4] MUELLER, K., ENGESSER, R., TIMMER, J., ZURBRIGGEN, M. D., Weber W.: Orthogonal Optogenetic Triple-Gene Control in Mammalian Cells. ACS Synth. Biol., Article ASAP<br></small>
 
[4] MUELLER, K., ENGESSER, R., TIMMER, J., ZURBRIGGEN, M. D., Weber W.: Orthogonal Optogenetic Triple-Gene Control in Mammalian Cells. ACS Synth. Biol., Article ASAP<br></small>
  
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== More Biology and Usage ==
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[http://2015.igem.org/Team:Valencia_UPV Valencia UPV team 2015] requested this part to the Registry. This part consists of the engineered PDZ domain, a crucial part of the light-inducible expression system. We designed, sequence synthetized and domesticated the AsLOV protein ([https://parts.igem.org/Part:BBa_K1742000 BBa_K1742000]) in order to complete this light-dependant switch system. The AsLOV domain is tagged with a small peptide in the C-terminus, which allows its recognition by ePDZ upon illumination with blue light. We used both parts to test their functionality as a light-inducible switch in ''Nicotiana benthamiana'' plants.
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In order to see if our blue light-dependant switch was functional in plants, the gene of interest assembled (GoldenBraid assembly) with the chimeric promoter (composed by the DBD operator ‘OpLacIBD’, a minimal promoter ‘miniP35S’ and a 5’-UTR region from TMV) was the Luciferase. In order to have a control for the Luciferase assay, we needed another construct already available in the GoldenBraid collection. It included the Renilla and P19 genes.
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https://static.igem.org/mediawiki/2015/thumb/5/53/BBa_K1742000_AsLOV_Luc_AssayFIXED.png/703px-BBa_K1742000_AsLOV_Luc_AssayFIXED.png
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<small><p><b>Figure 2. Luciferase assay showing the luciferase/renilla ratios. </b></p></small>
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'''References'''<br>
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<small>
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1. Zhang K., Cui., B (2015). Optogenetic control of intracellular signaling pathways. Trends in Biotechnology. 33: 92-100<br>
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2. Levskaya A., Weiner OD., Lim WA. Voigt CA (2009). Spatiotemporal control of cell signaling using a light-switchable protein interaction. Nature 461: 997-1001<br>
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3. Strickland D, Lin Y, Wagner E, Hope CM, Zayner J, Antoniou C, Sosnick TR, Weiss EL, Glotzer M (2012). TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat Methods 9:379-384<br>
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</small>
  
 
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Latest revision as of 04:52, 18 September 2015

engineered PDZ domain (ePDZ)

Usage and Biology

The engineered PDZ domain is a globuar protein domain of 80 - 90 amino acids wich has an enhanced interaction with other proteins. Normally PDZ domains bind to a short region at the C-terminus of other proteins but there are some proteins which recognize internal sequence motifs of target proteins through a single binding site on the domains [1][2]. There are more over 1000 known proteins containing this domain in eucaryotes and bacteria but just a few examples are shown in archea [3].
ePDZ is a crucial part of the blue light-inducible expression system. It is able to bind to the J-alpha-helix of the LOV2 domain. The principle of the light-induced expression is explained here [4].


We obtained the ePDZ from Konrad Müller, AG Weber of the University of Freiburg. It was stardardized and biobricked by us.

Figure 4: Efficiency of the blue light system using different time intervals of illumination. To test the efficiency of the light system, SEAP expression after illumination was determined. The SEAP assay was performed 24 hours after light exposure. Cells were incubated with blue light for 1 hour, 2.5 hours and 5 hours.

More information

References

[1] Cowburn D (December 1997). „Peptide recognition by PTB and PDZ domains“. Curr. Opin. Struct. Biol. 7 (6): 835–838
[2] Giallourakis C, Cao Z, Green T, Wachtel H, Xie X, Lopez-Illasaca M, Daly M, Rioux J, Xavier R. A molecular-properties-based approach to understanding PDZ domain proteins and PDZ ligands. Genome Res. 2006;16:1056–1072.
[3] Doyle DA, Lee A, Lewis J, Kim E, Sheng M, MacKinnon R (June 1996). „Crystal structures of a complexed and peptide-free membrane protein-binding domain: molecular basis of peptide recognition by PDZ“. Cell 85 (7): 1067–1076
[4] MUELLER, K., ENGESSER, R., TIMMER, J., ZURBRIGGEN, M. D., Weber W.: Orthogonal Optogenetic Triple-Gene Control in Mammalian Cells. ACS Synth. Biol., Article ASAP


More Biology and Usage

[http://2015.igem.org/Team:Valencia_UPV Valencia UPV team 2015] requested this part to the Registry. This part consists of the engineered PDZ domain, a crucial part of the light-inducible expression system. We designed, sequence synthetized and domesticated the AsLOV protein (BBa_K1742000) in order to complete this light-dependant switch system. The AsLOV domain is tagged with a small peptide in the C-terminus, which allows its recognition by ePDZ upon illumination with blue light. We used both parts to test their functionality as a light-inducible switch in Nicotiana benthamiana plants.

In order to see if our blue light-dependant switch was functional in plants, the gene of interest assembled (GoldenBraid assembly) with the chimeric promoter (composed by the DBD operator ‘OpLacIBD’, a minimal promoter ‘miniP35S’ and a 5’-UTR region from TMV) was the Luciferase. In order to have a control for the Luciferase assay, we needed another construct already available in the GoldenBraid collection. It included the Renilla and P19 genes.

703px-BBa_K1742000_AsLOV_Luc_AssayFIXED.png

Figure 2. Luciferase assay showing the luciferase/renilla ratios.

References
1. Zhang K., Cui., B (2015). Optogenetic control of intracellular signaling pathways. Trends in Biotechnology. 33: 92-100
2. Levskaya A., Weiner OD., Lim WA. Voigt CA (2009). Spatiotemporal control of cell signaling using a light-switchable protein interaction. Nature 461: 997-1001
3. Strickland D, Lin Y, Wagner E, Hope CM, Zayner J, Antoniou C, Sosnick TR, Weiss EL, Glotzer M (2012). TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat Methods 9:379-384

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 424
  • 1000
    COMPATIBLE WITH RFC[1000]