Difference between revisions of "Part:BBa K3064008"

 
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PGK promoter is a universal promoter which has a strong activation function and it has high content of G and C. PGK promoter is more effective than CMV in some tissue cells such as striatal cells and it is universally expressed in most organizations. PGK promoter is often used as a strong promoter to initiate the transcription of exogenous genes. People insert PGK promoter into vector to drive the expression of downstream target genes.
 
PGK promoter is a universal promoter which has a strong activation function and it has high content of G and C. PGK promoter is more effective than CMV in some tissue cells such as striatal cells and it is universally expressed in most organizations. PGK promoter is often used as a strong promoter to initiate the transcription of exogenous genes. People insert PGK promoter into vector to drive the expression of downstream target genes.
  
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===Usage and Biology===
 
===Usage and Biology===
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===Experiment===
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We inserted PGK promoter into plasmid pmirGLO and the promoter is upstream of the gene of fire luciferase.
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We laid cells, HepG2, in a 24-hole plate. When the confluence reached 70-90, pmirGLO plasmid was transfected. And then, culture media were divided into six groups of sugar concentration: 0mM, 5mM, 10mM, 15mM, 20mM, 30mM and were used to stimulate cells. After that, the cells were lysed and tested with a double luciferase kit. We found that expression of luciferase had significant changes with different sugar concentration.
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The extracellular sugar concentration in human physiological environment is about 22mM. And the experimental data showed that firefly luciferase gene which is downstream of PGK promoter expressed better and better when the sugar concentration is gradually approaching the physiological sugar concentration 22mM.
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https://static.igem.org/mediawiki/parts/f/f3/T--NUDT_CHINA--2019PicforpartPGK-1.jpg
  
 
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Revision as of 12:24, 15 October 2019


PGK promoter

PGK promoter is a universal promoter which has a strong activation function and it has high content of G and C. PGK promoter is more effective than CMV in some tissue cells such as striatal cells and it is universally expressed in most organizations. PGK promoter is often used as a strong promoter to initiate the transcription of exogenous genes. People insert PGK promoter into vector to drive the expression of downstream target genes.

Usage and Biology

Experiment

We inserted PGK promoter into plasmid pmirGLO and the promoter is upstream of the gene of fire luciferase.

We laid cells, HepG2, in a 24-hole plate. When the confluence reached 70-90, pmirGLO plasmid was transfected. And then, culture media were divided into six groups of sugar concentration: 0mM, 5mM, 10mM, 15mM, 20mM, 30mM and were used to stimulate cells. After that, the cells were lysed and tested with a double luciferase kit. We found that expression of luciferase had significant changes with different sugar concentration.

The extracellular sugar concentration in human physiological environment is about 22mM. And the experimental data showed that firefly luciferase gene which is downstream of PGK promoter expressed better and better when the sugar concentration is gradually approaching the physiological sugar concentration 22mM. T--NUDT_CHINA--2019PicforpartPGK-1.jpg

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 246
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 246
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 246
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 246
    Illegal AgeI site found at 111
  • 1000
    COMPATIBLE WITH RFC[1000]