Difference between revisions of "Part:BBa K2036021"
 
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CII ([https://parts.igem.org/Part:BBa_K2036000 BBa_K2036000]) functions as a transcriptional activator to direct promoter RE, so we constructed CII-TT-pRE-RBS-GFP-LVAssrAtag as test group and pRE-RBS-GFPLVAssrAtag as CK to see if CII efficiently activate pRE.
 
CII ([https://parts.igem.org/Part:BBa_K2036000 BBa_K2036000]) functions as a transcriptional activator to direct promoter RE, so we constructed CII-TT-pRE-RBS-GFP-LVAssrAtag as test group and pRE-RBS-GFPLVAssrAtag as CK to see if CII efficiently activate pRE.
 
</p>
 
</p>
−
[[File: T--HUST-China--CII-pRE_plate.png |thumb|800px|center|Fig: CII and pRE activation test]]
+
[[File: T--HUST-China--CII-pRE_plate.png |thumb|800px|center|Fig2: CII and pRE activation test]]
 
<p>
 
<p>
 
According to the Flourescence measurement curve above, we can see clearly that GFP level increased over time and it showed significant difference from CK.
 
According to the Flourescence measurement curve above, we can see clearly that GFP level increased over time and it showed significant difference from CK.
 
We also did Fluorescence microscope detection after 30, 120 and 240 minutes induction. According to the figture below, we can tell qualitively that pRE leakage are at relative low level and CII can efficiently activate the promoter.
 
We also did Fluorescence microscope detection after 30, 120 and 240 minutes induction. According to the figture below, we can tell qualitively that pRE leakage are at relative low level and CII can efficiently activate the promoter.
 
</p>
 
</p>
−
[[File: T--HUST-China--Experiments-CII-pRE_Flou-detec.png|thumb|800px|center|Fig: Fluorescence detection]]
+
[[File: T--HUST-China--Experiments-CII-pRE_Flou-detec.png|thumb|800px|center|Fig3: Fluorescence detection]]
 
<br>
 
<br>
 
<p>
 
<p>
 
We characterized cro and pRM inhibition by the same method as CI and pR’s. From line chart and fluorescence detection, we can see that the test group contains cro expressed less GFP protein than control group over time. It proves that cro can effectively bind pRM to block its downstream gene’s transcription.
 
We characterized cro and pRM inhibition by the same method as CI and pR’s. From line chart and fluorescence detection, we can see that the test group contains cro expressed less GFP protein than control group over time. It proves that cro can effectively bind pRM to block its downstream gene’s transcription.
 
</p>
 
</p>
−
[[File: T--HUST-China--CI-pR_inhibition.png |thumb|800px|center|Fig: Cro and pRM inhibition test]]
+
[[File: T--HUST-China--CI-pR_inhibition.png |thumb|800px|center|Fig4: Cro and pRM inhibition test]]
 
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Latest revision as of 05:22, 25 October 2016


placm-pRE-RBS-Cro-RBS-CII-TT

In order to test Signal Filter in real condition, we constructed an application plasmid applying to lactose intorlerance （see to Fig1）. And because the circuit is complex, we employed In-Fusion cloning method to build its subparts,this is one of them.

Fig1:Application of Signal Filter expression plasmid

Protein&promoter

CII (BBa_K2036000) functions as a transcriptional activator to direct promoter RE, so we constructed CII-TT-pRE-RBS-GFP-LVAssrAtag as test group and pRE-RBS-GFPLVAssrAtag as CK to see if CII efficiently activate pRE.

Fig2: CII and pRE activation test

According to the Flourescence measurement curve above, we can see clearly that GFP level increased over time and it showed significant difference from CK. We also did Fluorescence microscope detection after 30, 120 and 240 minutes induction. According to the figture below, we can tell qualitively that pRE leakage are at relative low level and CII can efficiently activate the promoter.

Fig3: Fluorescence detection


We characterized cro and pRM inhibition by the same method as CI and pR’s. From line chart and fluorescence detection, we can see that the test group contains cro expressed less GFP protein than control group over time. It proves that cro can effectively bind pRM to block its downstream gene’s transcription.

Fig4: Cro and pRM inhibition test


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]