We designed <h4>[https://parts.igem.org/Part:BBa_K2986004 Biobrick BBa_K2086004]</h4> rather than optimizing Gluc for usage 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.
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We designed <h4>[https://parts.igem.org/Part:BBa_K2986004 Biobrick BBa_K2086004, hGluc]</h4> as a improvement for the Gluc to the humanized Gaussia princeps which can be expressed in human cell lines.
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<h2>Design</h2>
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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.
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===Location of features===
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[[File:T--SUSTech-enzyme.png|400px|thumb|center|Figure1.the plasmid used with hgluc]]
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hGluc: 3120-3710<br/>
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linker: 3711-3755<br/>
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CD11b transmembrane domain: 3756-3989<br/>
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P2A :3990-4055<br/>
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Interleukin 8 CDS :4056-4352<br/>
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<h2>Properties</h2>
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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.
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And we tested the fluorescent activity under different light exposure time, and analyzed data is shown here.
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[[File:T--SUSTech hGluc.png|450px|thumb|center|Figure2.hgluc activity after light exposure ]]
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We characterized the transcription process by testing the change of RNA through quantitative PCR. Next, we characterized the translation process by testing the dynamic change of mRuby through flow cytometer. The final step is to characterize the secretion process. Since we have chosen hGluc as our target product, we did it by measuring the chemiluminescence value.
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[[File:T--SUSTech--yong13.png|400px|thumb|center|Figure3. Result of hGluc chemiluminescence value on secretion characterization]]
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For the hGluc chemiluminescence test, we characterized the secretion process after protein translation. This set of data enable us to characterize he relationship between light exposure and Gene expression on multi-level (transcription level, translation level and secretion level), which is vital for further acquisition of experimental parameters and model constructions.
Latest revision as of 21:08, 21 October 2019
GLuc
Usage and Biology
This is a Bioluminescent luciferase protein that emits green light, and so can be used as a reporter. It was isolated from the sea creature Gaussia princeps. This version of the gene has been optimised for use in the algal species Chlamydomonas reinhardtii.
