Difference between revisions of "Part:BBa K2036000"
 
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<partinfo>BBa_K2036000 short</partinfo>
 
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CII is an important part of the bacteriaphage lambda operon. It promotes lysogeny through activation of three phage promoters p(E), p(I) and p(aQ), recognizing a direct repeat sequence TTGCN6TTGC at each. It is an unstable protein in vivo, being rapidly degraded by the host protease HflB (FtsH). This instability is essential for the function of CII in the lysis-lysogeny switch. From NMR and limited proteolysis we show that about 15 C-terminal residues of CII are highly flexible, and may act as a target for proteolysis in vivo. From in vitro transcription, isothermal calorimetry and gel chromatography of CII (1-97) and its truncated fragments CIIA (4-81/82) and CIIB (4-69), we find that residues 70-81/82 are essential for (a) tetramer formation, (b) operator binding and (c) transcription activation. Presumably, tetramerization is necessary for the latter functions.
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CII is an important part of the bacteriophage lambda operon. It promotes lysogeny through activation of three phage promoters p(E), p(I) and p(aQ), recognizing a direct repeat sequence TTGCN6TTGC at each. It is an unstable protein in vivo, being rapidly degraded by the host protease HflB (FtsH). This instability is essential for the function of CII in the lysis-lysogeny switch. From NMR and limited proteolysis we show that about 15 C-terminal residues of CII are highly flexible, and may act as a target for proteolysis in vivo. From in vitro transcription, isothermal calorimetry and gel chromatography of CII (1-97) and its truncated fragments CIIA (4-81/82) and CIIB (4-69), we find that residues 70-81/82 are essential for (a) tetramer formation, (b) operator binding and (c) transcription activation. Presumably, tetramerization is necessary for the latter functions.
  
 
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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.
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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-GFP-LVAssrAtag as CK to see if CII efficiently activate pRE.
 
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[[File:T--HUST-China--CII-pRE_plate.png|800px|thumb|center|Fig1: According to the Flourescence measurement curve above, we can see clearly that GFP level increasedover time and it showed significant difference from CK.
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[[File:T--HUST-China--CII-pRE_plate.png|800px|thumb|center|Fig1: 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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[[File:T--HUST-China--Experiments-CII-pRE_Flou-detec.png|800px|thumb|center|Fig2: 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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[[File:T--HUST-China--Experiments-CII-pRE_Flou-detec.png|800px|thumb|center|Fig2: We also did Fluorescence microscope detection after 30, 120 and 240 minutes induction.According to the figure below, we can tell qualitively that pRE leakage is at relatively low level and CII can efficiently activate the promoter.
 
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<h2>Protein&protein reaction</h2>
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We had submitted and documented RBS-CIII-RBS-CIII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag ([https://parts.igem.org/Part:BBa_K2036014 BBa_K2036014]) and RBS-CII-RBS-CII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag ([https://parts.igem.org/Part:BBa_K2036015 BBa_K2036015]). These two parts were to test whether CIII can protect CII from being degraded by Ftsh by competitive inhibition.
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[[File:T--HUST-China--CIII%26Ftsh.png|800px|thumb|center|Fig3: According to the flourescence measurement curve above, we can see clearly that GFP level of CIII test circuit increased over time and it showed significant difference from two control groups. It indicates that tandemly expressed CIII can efficiently protect CII from being degraded by Ftsh. ]]

Latest revision as of 05:17, 25 October 2016


CII bacteriophage lambda trascriptional activator

CII is an important part of the bacteriophage lambda operon. It promotes lysogeny through activation of three phage promoters p(E), p(I) and p(aQ), recognizing a direct repeat sequence TTGCN6TTGC at each. It is an unstable protein in vivo, being rapidly degraded by the host protease HflB (FtsH). This instability is essential for the function of CII in the lysis-lysogeny switch. From NMR and limited proteolysis we show that about 15 C-terminal residues of CII are highly flexible, and may act as a target for proteolysis in vivo. From in vitro transcription, isothermal calorimetry and gel chromatography of CII (1-97) and its truncated fragments CIIA (4-81/82) and CIIB (4-69), we find that residues 70-81/82 are essential for (a) tetramer formation, (b) operator binding and (c) transcription activation. Presumably, tetramerization is necessary for the latter functions.

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]


Protein&promoter

--CII and pRE


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.

Fig1: According to the flourescence measurement curve above, we can see clearly that GFP level increased over time and it showed significant difference from CK.
Fig2: We also did Fluorescence microscope detection after 30, 120 and 240 minutes induction.According to the figure below, we can tell qualitively that pRE leakage is at relatively low level and CII can efficiently activate the promoter.

Protein&protein reaction

We had submitted and documented RBS-CIII-RBS-CIII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036014) and RBS-CII-RBS-CII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036015). These two parts were to test whether CIII can protect CII from being degraded by Ftsh by competitive inhibition.


Fig3: According to the flourescence measurement curve above, we can see clearly that GFP level of CIII test circuit increased over time and it showed significant difference from two control groups. It indicates that tandemly expressed CIII can efficiently protect CII from being degraded by Ftsh.