Difference between revisions of "Part:BBa K509005"

Revision as of 03:10, 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.

Improvement from SUSTech_Shenzhen 2019

We designed

Biobrick BBa_K2086004

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.

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

Figure1.the plasmid used with hgluc

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.

Figure2.hgluc activity after light exposure

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.

Figure3. Result of hGluc chemiluminescence value on secretion characterization

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.

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 NgoMIV site found at 205
Illegal NgoMIV site found at 550
1000
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K509005 short</partinfo>
-===Usage and Biology===
+<h2>Usage and Biology</h2>
 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''.
 <h2>Improvement from SUSTech_Shenzhen 2019</h2>
 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.
 <h2>Design</h2>