Difference between revisions of "Part:BBa K3064026"
(→‎Characterization)
Line 19: Line 19:
  
 
===Characterization===
 
===Characterization===
−
For characterizaton, this part BBa_K3064026 was cloned in pcDNA3.1+ and transfected into HepG2 cell lines using Invitrogen LipofectamineTM 3000. Protocol could be found in our Experiment page. After transfection 12h, we starve the HepG2 cells with glucose-free culture and stimulate with 20mM glucose concentration culture after 6 more hours. Red fluorescence protein mCherry is used to present the promoter intensity at different time. After glucose stimulation of 6, 18, 30 and 50 hours we took photos of cells with fluorescence microscopy and calculated grey level. Here is the result.  
+
For characterizaton, this part BBa_K3064026 was cloned in pcDNA3.1+ and transfected into HepG2 cell lines using Invitrogen LipofectamineTM 3000. Protocol could be found in our Experiment page. After transfection 12h, we starve the HepG2 cells with glucose-free culture and stimulate with 20mM glucose concentration culture after 6 more hours. Glucose stimulation intensity controlled at 20mM. Sample tested after transfection of 48h.
  
−
https://static.igem.org/mediawiki/parts/4/4d/T--NUDT_CHINA--silver-3.jpg
+
https://static.igem.org/mediawiki/parts/3/3c/T--NUDT_CHINA--gold.jpg
  
−
 
+
Figure 1. Firefly luciferase/renilla luciferase RLU for minipromoter and 3/6/9 glucose sensing promoter.
−
 
+
−
It is obvious that from 6h to 38h after glucose stimulation the
+

Revision as of 02:33, 19 October 2019


9xGSP-GFP

This composite part is made up of a kind of improved promoter which is sensitive to particular high blood glucose concentration and EGFP sequence. The introduction of EGFP makes it possible to examine the expression of this composite part. As designed,the part can be used as a glucose-sensing promoter with GFP reporter system.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

For characterizaton, this part BBa_K3064026 was cloned in pcDNA3.1+ and transfected into HepG2 cell lines using Invitrogen LipofectamineTM 3000. Protocol could be found in our Experiment page. After transfection 12h, we starve the HepG2 cells with glucose-free culture and stimulate with 20mM glucose concentration culture after 6 more hours. Glucose stimulation intensity controlled at 20mM. Sample tested after transfection of 48h.

T--NUDT_CHINA--gold.jpg

Figure 1. Firefly luciferase/renilla luciferase RLU for minipromoter and 3/6/9 glucose sensing promoter.