Difference between revisions of "Part:BBa K2986004"
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The luciferase from the marine copepod Gaussia princeps (Gluc) is the smallest coelenterazine (CTZ)-utilizing luciferase known thus far. It can emit green light and this property makes it to be used as a reporter gene. Gaussia luciferase exhibits an activity up to 1,000-fold higher than to Renilla reniformis luciferase (Rluc), firefly luciferase (Fluc), or bacterial luciferases (LuxAB). The outstanding sensitivity of Gluc-based assays was previously demonstrated detecting as low as 10−18 mol purified Gluc or one eukaryotic cell transiently expressing Gluc. | The luciferase from the marine copepod Gaussia princeps (Gluc) is the smallest coelenterazine (CTZ)-utilizing luciferase known thus far. It can emit green light and this property makes it to be used as a reporter gene. Gaussia luciferase exhibits an activity up to 1,000-fold higher than to Renilla reniformis luciferase (Rluc), firefly luciferase (Fluc), or bacterial luciferases (LuxAB). The outstanding sensitivity of Gluc-based assays was previously demonstrated detecting as low as 10−18 mol purified Gluc or one eukaryotic cell transiently expressing Gluc. | ||
| − | We decided to modify the [https://parts.igem.org/Part:BBa_K509005 Biobrick BBa_K509005] from iGEM11_UEA-JIC_Norwich 2011 team, this team optimized Gluc for use in the algal species Chlamydomonas reinhardtii. We designed to improve the Gluc to the humanized Gaussia princeps which can be expressed in mammalian cells. This made the visualization of the target gene expression possible if we ligated the hGluc with target gene. | + | We decided to modify the [https://parts.igem.org/Part:BBa_K509005 Biobrick BBa_K509005]from iGEM11_UEA-JIC_Norwich 2011 team, this team optimized Gluc for use in the algal species Chlamydomonas reinhardtii. We designed to improve the Gluc to the humanized Gaussia princeps which can be expressed in mammalian cells. This made the visualization of the target gene expression possible if we ligated the hGluc with target gene. |
[[File:The_plasmid_with_il8_and_hgluc.png|450px|thumb|center|Figure1.the plasmid used with hgluc]] | [[File:The_plasmid_with_il8_and_hgluc.png|450px|thumb|center|Figure1.the plasmid used with hgluc]] | ||
<h1>Design</h1> | <h1>Design</h1> | ||
| − | We wanted to design a gene expression system with hGluc as reporter, so that we can simplify the production detection process into fluorescent observation. We designed a plasmid ligate our target gene with hGluc using mammalian lentivirus expression vector. We use a 45bp linker to connect the hGluc with the CD11b transmembrane domain and use P2A connect with Interleukin 8 CDS, in our assumption this allow the stable expression of both our target gene and the hGluc in Hela cells.<br/> | + | We wanted to design a gene expression system with hGluc as reporter, so that we can simplify the production detection process into fluorescent observation. We designed a plasmid ligate our target gene with hGluc using mammalian lentivirus expression vector. We use a 45bp linker to connect the hGluc with the CD11b transmembrane domain and use P2A (2A peptide allows an open reading frame (ORF) to translate a peptide chain into several independent peptide chains) connect with Interleukin 8 CDS, in our assumption this allow the stable expression of both our target gene and the hGluc in Hela cells.<br/> |
===Location of features=== | ===Location of features=== | ||
hGluc: 3120-3710<br/> | hGluc: 3120-3710<br/> | ||
linker: 3711-3755<br/> | linker: 3711-3755<br/> | ||
CD11b transmembrane domain: 3756-3989<br/> | CD11b transmembrane domain: 3756-3989<br/> | ||
| − | P2A | + | P2A :3990-4055<br/> |
Interleukin 8 CDS :4056-4352<br/> | Interleukin 8 CDS :4056-4352<br/> | ||
Revision as of 05:20, 19 October 2019
hGluc
Usage and Biology
The luciferase from the marine copepod Gaussia princeps (Gluc) is the smallest coelenterazine (CTZ)-utilizing luciferase known thus far. It can emit green light and this property makes it to be used as a reporter gene. Gaussia luciferase exhibits an activity up to 1,000-fold higher than to Renilla reniformis luciferase (Rluc), firefly luciferase (Fluc), or bacterial luciferases (LuxAB). The outstanding sensitivity of Gluc-based assays was previously demonstrated detecting as low as 10−18 mol purified Gluc or one eukaryotic cell transiently expressing Gluc. We decided to modify the Biobrick BBa_K509005from iGEM11_UEA-JIC_Norwich 2011 team, this team optimized Gluc for use in the algal species Chlamydomonas reinhardtii. We designed to improve the Gluc to the humanized Gaussia princeps which can be expressed in mammalian cells. This made the visualization of the target gene expression possible if we ligated the hGluc with target gene.
Design
We wanted to design a gene expression system with hGluc as reporter, so that we can simplify the production detection process into fluorescent observation. We designed a plasmid ligate our target gene with hGluc using mammalian lentivirus expression vector. We use a 45bp linker to connect the hGluc with the CD11b transmembrane domain and use P2A (2A peptide allows an open reading frame (ORF) to translate a peptide chain into several independent peptide chains) connect with Interleukin 8 CDS, in our assumption this allow the stable expression of both our target gene and the hGluc in Hela cells.
Location of features
hGluc: 3120-3710
linker: 3711-3755
CD11b transmembrane domain: 3756-3989
P2A :3990-4055
Interleukin 8 CDS :4056-4352
Properties
Later we transfected this plasmid into Hela cells, we tested whether we successfully transfected. We use the hGluc kit to test the function of hGluc after assembled into the plasmid. The result showed that hGluc can normally paly its role. And we tested the fluorescent activity under different light exposure time, and analyzed data is shown here.
References
Tannous B. A. (2009). Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. Nature protocols, 4(4), 582–591. doi:10.1038/nprot.2009.28.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 604