Difference between revisions of "Part:BBa K2036018"
 
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<partinfo>BBa_K2036018 short</partinfo>
 
<partinfo>BBa_K2036018 short</partinfo>
  
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This is a part of Prokaryote version of Signal Filter characterization circuit:pRE-RBS-Cro-RBS-CII-TT-ptrp-RBS-CI-TT-pR-RBS-CIII-RBS-RFP-LAAssrAtag-TT-pRM-RBS-GFP-LVAssrAtag ([https://parts.igem.org/Part:BBa_K2036027 BBa_K2036027]).
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This is a part of Prokaryote version of Signal Filter characterization([http://2016.igem.org/Team:HUST-China Details see to HUST-China 2016 wiki])  circuit:pRE-RBS-Cro-RBS-CII-TT-ptrp-RBS-CI-TT-pR-RBS-CIII-RBS-RFP-LAAssrAtag-TT-pRM-RBS-GFP-LVAssrAtag ([https://parts.igem.org/Part:BBa_K2036027 BBa_K2036027]).
 
[[File:T--HUST-China--Experiments-Fig14-1.png|thumb|500px|center|Fig1:Prokaryote version of Signal Filter characterization circuit]]
 
[[File:T--HUST-China--Experiments-Fig14-1.png|thumb|500px|center|Fig1:Prokaryote version of Signal Filter characterization circuit]]
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<br>Because the circuit is too complex we divided it into three parts and each is assembled by In-Fusion method (homologous recombination). The whole circuit built by 3A assembly and then we transfer the backbone from pSB1C3 to PET-Duet-1.  
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<br>Because the circuit is too complex, we divided it into three parts and each is assembled by In-Fusion method (homologous recombination). The whole circuit is built by 3A assembly and then we transfer the backbone from pSB1C3 to PET-Duet-1.  
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[[File:T--HUST-China--Experiments-Fig13.png|thumb|500px|center|Fig2:Prokaryote version of Signal Filter characterization plasmid]]
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[[File:T--HUST-China--Experiments-Fig13.png|thumb|350px|center|Fig2:Prokaryote version of Signal Filter characterization expression plasmid]]
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<p>
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CII ( [https://parts.igem.org/Part:BBa_K2036000 BBa_K2036000]
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) functions as a transcriptional activator to direct promoter RE, so we constructed CII-TT-pRE-RBS-GFP-LVAssrAtag as test group and pRE-RBS-GFP-LVAssrAtag as CK to see if CII efficiently activate pRE.
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</p>
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<br>
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[[File:T--HUST-China--CII-pRE_plate.png|thumb|800px|center|Fig3: CII and pRE activation test]]
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<p>
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According to the flourescence measurement curve above, we can see clearly that GFP level increased over time and it showed significant difference from CK.
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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.
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</p>
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<br>
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[[File:T--HUST-China--Experiments-CII-pRE_Flou-detec.png|thumb|800px|center|Fig4: Fluorescence detection]]
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<h3>Preliminary experiments of ptrp2</h3>
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<p>
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ptrp2( [https://parts.igem.org/Part:BBa_K203601324 BBa_K2036024]
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) is an improved part from HUST-China 2015, we employed it as one of our signal sensor to test our tri-stable switch. We constructed ptrp2-GFP-pSB1C3 to determine an appropriate induction concentration.
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</p>
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[[File:T--HUST-China--ptrp-IAA.png|thumb|800px|center|Fig5:According to the GFP expression curve, IAA induction of ptrp2 with 50μM final concentration is a better choice relatively.]]
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 06:34, 25 October 2016


pRE-RBS-Cro-RBS-CII-TT-ptrp2

This is a part of Prokaryote version of Signal Filter characterization([http://2016.igem.org/Team:HUST-China Details see to HUST-China 2016 wiki]) circuit:pRE-RBS-Cro-RBS-CII-TT-ptrp-RBS-CI-TT-pR-RBS-CIII-RBS-RFP-LAAssrAtag-TT-pRM-RBS-GFP-LVAssrAtag (BBa_K2036027).

Fig1:Prokaryote version of Signal Filter characterization circuit


Because the circuit is too complex, we divided it into three parts and each is assembled by In-Fusion method (homologous recombination). The whole circuit is built by 3A assembly and then we transfer the backbone from pSB1C3 to PET-Duet-1.

Fig2:Prokaryote version of Signal Filter characterization expression plasmid

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-GFP-LVAssrAtag as CK to see if CII efficiently activate pRE.


Fig3: 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.


Fig4: Fluorescence detection

Preliminary experiments of ptrp2

ptrp2( BBa_K2036024 ) is an improved part from HUST-China 2015, we employed it as one of our signal sensor to test our tri-stable switch. We constructed ptrp2-GFP-pSB1C3 to determine an appropriate induction concentration.

Fig5:According to the GFP expression curve, IAA induction of ptrp2 with 50μM final concentration is a better choice relatively.


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]