Difference between revisions of "Part:BBa K2865008"

Latest revision as of 17:01, 13 October 2018

BNP promoter-eGFP-poly(A)

This is a mammalian eGFP generator used for characterization of our BNP promoter (BBa_K2865000). This eGFP generator contains the BNP promoter sequence followed by eGFP reporter in RFC25 format(BBa_K2865005) and SV40 polyA terminator (BBa_K2865004). This eGFP generator was then transfected into H9C2 cells and in vivo green fluorescence signal was observed under fluorescence microscope.

Fig.1 The construction of part

Experiment

Fluorescence microscope

We transfected H9C2 cell with this part by liposome transfection. And we select two widely used chemicals to simulate cell microenvironment in heart failure, ET1 and Ang II. From scientists’ previous work, we learnt that the appropriate concentration is 10^-7mol/L and BNP is in really high expression in 10^-7mol/L.

After 48h of expression, we detected all the cells using Fluorescence microscope. However, the BNP promoter did not work very well. All the groups using ET1 and AngII have no green fluorescent signal. At the same time, the groups using CMV promoter succeeded in expressing eGFP.

Figure.2 Expression of BNP-eGFP 48 h after the administration of AngⅡand ET-1 under fluorescent microscope. (A) was without adding. (B) was cells treated with 10^-7 mol/L AngⅡ and (C) was cells treated with 10^-7mol/L ET-1.

Figure.3 Expression of CMV-eGFP 48 h after the administration of AngⅡand ET-1 under fluorescent microscope. (A) was without adding. (B) was cells treated with 10^-7 mol/L AngⅡ and (C) was cells treated with 10^-7mol/L ET-1.

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 1284
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 242

@@ Line 9: / Line 9: @@
 <p class="MsoNormal"><span lang="EN-US">We transfected H9C2 cell with this part by liposome transfection. And we select two widely used chemicals to simulate cell microenvironment in heart failure, ET1 and Ang II. From scientists’ previous work, we learnt that the appropriate concentration is 10<sup>-7</sup>mol/L and BNP is in really high expression in 10<sup>-7</sup>mol/L. </span></p>
 <p class="MsoNormal"><span lang="EN-US">After 48h of expression, we detected all the cells using Fluorescence microscope. However, the BNP promoter did not work very well. All the groups using ET1 and AngII have no green fluorescent signal. At the same time, the groups using CMV promoter succeeded in expressing eGFP. </span></p>
-[[File:T--SMMU-China--bep 2.png|550px|thumb|center|Figure.3 Expression of BNP-eGFP 48 h after the administration of AngⅡand ET-1 under fluorescent microscope. (A) was without adding. (B) was cells treated with 10<sup>-7</sup> mol/L AngⅡ and (C) was cells treated with 10<sup>-7</sup>mol/L ET-1.]]
+[[File:T--SMMU-China--bep 2.png|550px|thumb|center|Figure.2 Expression of BNP-eGFP 48 h after the administration of AngⅡand ET-1 under fluorescent microscope. (A) was without adding. (B) was cells treated with 10<sup>-7</sup> mol/L AngⅡ and (C) was cells treated with 10<sup>-7</sup>mol/L ET-1.]]
+[[File:T--SMMU-China--CMV et-1+Ang 2.png|550px|thumb|center|Figure.3 Expression of CMV-eGFP 48 h after the administration of AngⅡand ET-1 under fluorescent microscope. (A) was without adding. (B) was cells treated with 10<sup>-7</sup> mol/L AngⅡ and (C) was cells treated with 10<sup>-7</sup>mol/L ET-1.]]
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