Difference between revisions of "Part:BBa K2986004"

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−	===Usage and Biology===
−	The luciferase from the marine copepod Gaussia princeps (Gluc) is the smallest (&#8764;19.9-kDa) coelenterazine (CTZ)-utilizing luciferase known thus far.  A humanized variant of Gluc (hGluc), which was codon optimized for expression in cultured mammalian cells, has been widely 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&#8722;18 mol purified Gluc or one eukaryotic cell transiently expressing Gluc.
−	[[File:The_plasmid_with_il8_and_hgluc.png|450px|thumb|left|Figjre1.the plasmid used with hgluc]]	+	<h2>Usage and Biology</h2>
−	we use the plamid contains the hGluc, mRuby to be a quantity that characterize the the level of secretion the cytokine Il8.	+	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.<br/>
−	Interleukin 8 (IL8 or chemokine (C-X-C motif) ligand 8, CXCL8) is a chemokine produced by macrophages and other cell types such as epithelial cells, airway smooth muscle cells and endothelial cells. Endothelial cells store IL-8 in their storage vesicles, the Weibel-Palade bodies. In humans, the interleukin-8 protein is encoded by the CXCL8 gene. IL-8 is initially produced as a precursor peptide of 99 amino acids which then undergoes cleavage to create several active IL-8 isoforms.In culture, a 72 amino acid peptide is the major form secreted by macrophages.	+
−	There are many receptors on the surface membrane capable of binding IL-8; the most frequently studied types are the G protein-coupled serpentine receptors CXCR1 and CXCR2. Expression and affinity for IL-8 differs between the two receptors (CXCR1 > CXCR2). Through a chain of biochemical reactions, IL-8 is secreted and is an important mediator of the immune reaction in the innate immune system response.	+	We decided to modify the <h4>[https://parts.igem.org/Part:BBa_K509005 Biobrick BBa_K509005]from iGEM11_UEA-JIC_Norwich 2011 team</h4> 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 human cell lines.
−	IL-8 can be secreted by any cells with toll-like receptors that are involved in the innate immune response. Usually, it is the macrophages that see an antigen first, and thus are the first cells to release IL-8 to recruit other cells. Both monomer and homodimer forms of IL-8 have been reported to be potent inducers of the chemokine receptors CXCR1 and CXCR2. The homodimer is more potent, but methylation of Leu25 can block the activity of homodimers.
−	IL-10 is a cytokine with multiple, pleiotropic, effects in immunoregulation and inflammation. It downregulates the expression of Th1 cytokines, MHC class II antigens, and co-stimulatory molecules on macrophages. It also enhances B cell survival, proliferation, and antibody production. IL-10 can block NF-κB activity, and is involved in the regulation of the JAK-STAT signaling pathway.
−	Discovered in 1991 IL-10 was initially reported to suppress cytokine secretion, antigen presentation and CD4+ T cell activation. Further investigation has shown that IL-10 predominantly inhibits lipopolysaccharide (LPS) and bacterial product mediated induction of the pro-inflammatory cytokines TNFα, IL-1β, IL-12, and IFNγ secretion from Toll-Like Receptor (TLR) triggered myeloid lineage cells.	+	<h2>Design</h2>
		+	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.
−	===Experiment===
−	[[File:Hgluc with il8 flow.png|450px|thumb|left|Figjre2.hGluc flow and Il8 flow]]
−	[[File:HGlu secretion and IL8 elisa.png|450px|thumb|left|Figjre3.hGluc secretion and Il8 Elisa]]
−	We use the hGluc to be a quantity that characterize the the level of secretion the cytokine Interleukin 8. We can see from the curve that the tendency of the hGluc is similiar to the secertion of the Il8. This shows our plasmid works will in transfering the hGluc-bound cytokine expression system into the target cells.	+	===Location of features===
		+	[[File:T--SUSTech-enzyme.png|400px|thumb|center|Figure1.the plasmid used with hgluc]]
		+	hGluc: 3120-3710<br/>
		+	linker: 3711-3755<br/>
		+	CD11b transmembrane domain: 3756-3989<br/>
		+	P2A :3990-4055<br/>
		+	Interleukin 8 CDS :4056-4352<br/>
		+	<h2>Properties</h2>
		+	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.
		+	<html>
		+	<figure>
		+	<img src="https://2019.igem.org/wiki/images/c/cc/T--SUSTech_Shenzhen--cck8.jpg" "alt="" style="width:50%;" />
		+	<figcaption>hGluc chemiluminescence test</figcaption>
		+	</figure>
		+	</html>
		+	[[File:T--SUSTech hGluc.png|450px|thumb|center|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.
		+	[[File:T--SUSTech--yong13.png|400px|thumb|center|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.
		+	<h2>References</h2>
		+	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.
−
−	===Modeling===
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−	[[File:IL 10 data fitting parameter estimation.png|450px|thumb|left|Figjre4.IL 10 (102.4uw light intensity) data fitting & parameter estimation]]
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−	we set up a mathematic model to stimnulate the process of expressing the cytokine and Notation: X: monomer, X*: dimer, D: promoter, P: polymerase, 𝑌_𝑚: mRNA. Parameter: 𝑐_1: total # of X, 𝑐_2: total # of D, c: light density, a: top, b: bottom, 𝑛_1&𝑛_2: hill slope, 𝐾_1&𝐾_2: activation coefficient.
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−	[[File:The model.png|450px|thumb|left|Figjre5.The mathematic model]]
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−	</p>
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−	===Design Notes===
−	how to use the hGluc to represent the cytokine expression.
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−	===References===
−	[1].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.
−
−	[2].Hedges JC, Singer CA, Gerthoffer WT (2000). "Mitogen-activated protein kinases regulate cytokine gene expression in human airway myocytes". Am. J. Respir. Cell Mol. Biol. 23 (1): 86–94. CiteSeerX 10.1.1.326.6212. doi:10.1165/ajrcmb.23.1.4014. PMID 10873157.
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−	[3].Brat DJ, Bellail AC, Van Meir EG (2005). "The role of interleukin-8 and its receptors in gliomagenesis and tumoral angiogenesis". Neuro-oncology. 7 (2): 122–133. doi:10.1215/s1152851704001061. PMC 1871893. PMID 15831231.
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−	[4].Utgaard JO, Jahnsen FL, Bakka A, Brandtzaeg P, Haraldsen G (1998). "Rapid secretion of prestored interleukin 8 from Weibel-Palade bodies of microvascular endothelial cells". J. Exp. Med. 188 (9): 1751–6. doi:10.1084/jem.188.9.1751. PMC 2212514. PMID 9802986.
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−	[5].Modi WS, Dean M, Seuanez HN, Mukaida N, Matsushima K, O'Brien SJ (1990). "Monocyte-derived neutrophil chemotactic factor (MDNCF/IL-8) resides in a gene cluster along with several other members of the platelet factor 4 gene superfamily". Hum. Genet. 84 (2): 185–7. doi:10.1007/BF00208938. PMID 1967588.
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	<span class='h3bb'>Sequence and Features</span>		<span class='h3bb'>Sequence and Features</span>
	<partinfo>BBa_K2986004 SequenceAndFeatures</partinfo>		<partinfo>BBa_K2986004 SequenceAndFeatures</partinfo>

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Latest revision as of 21:26, 21 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 human cell lines.

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.

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.

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.

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