Difference between revisions of "Part:BBa K243032"
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<partinfo>BBa_K243032 short</partinfo>
 
<partinfo>BBa_K243032 short</partinfo>
  
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This part is like part the [https://parts.igem.org/Part:BBa_K243000 protein domain Fok_a] from our universal endonuclease, but it has an additional linked [https://parts.igem.org/Part:BBa_K157008 YFP-marker].   
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This part is composed of the essential Fok_a part [https://parts.igem.org/Part:BBa_K243000 protein domain Fok_a] of our universal endonuclease, but additionally linked to a [https://parts.igem.org/Part:BBa_K157008 Venus-marker] to detect even low levels of protein expression by fluorescence microscopy.   
  
 
===Usage and Biology===
 
===Usage and Biology===
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The addition of a fluorescent protein(Venus) to the protein construct, enables the detection by fluorescence microscope and new way of purification by GFP-trap column.The Venus protein was fusioned C'terminal to the protein domain, so when the protein is expressed the fluorescence signal of the Venus protein can be seen.
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The addition of a fluorescent protein (Venus) to the protein construct enables easy detection of Fok_a expression by fluorescence microscopy as well as a new way of purification by a GFP-trap column. The Venus protein was C-terminally fused to the Fok_a protein domain. In the opposite orientation, skipped expression of Fok_a would not be detectable. When the fusion protein is expressed by E. coli, the fluorescence signal of the Venus protein can be detected. In addition to these methods, anti-GFP antibodies can be used to check both the protein size and presence by western blotting.
 
<br>
 
<br>
 
===Detection===
 
===Detection===
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There was a strong fluorescence signal under the fluorescence microscope after the induction of the cells with IPTG.The ''E.colis''(RV308) were excited with the wavelength 505nm and then some pictures of the fluorescencewere made.<br>
 
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[[Image:Freiburg RV308 mit fluo Oligos3 (c1+c2).JPG|350x250px]]
 
  
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===Purification===
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A strong fluorescence signal could be detected using the fluorescence microscope and IPTG-induced cells. ''E. coli''(RV308) were excited by light of the wavelength 505 nm and fluorescence pictures were made using a cutoff filter.<br>
  
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[[Image:FokA Venus.JPG|500x500px]]<br>
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The cell extracts were checked by SDS-page to get information about the expression of proteins.<br><br>
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[[Image:SDSfokvenus.jpg]]<br>
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SDS gel; pEx Venus-Fok_a in BL21de3; lanes: Marker (ColorPlus Prestained Protein Marker, Broad Range), clone 1 uninduced, clone 1 induced, clone 1 induced, clone 2 uninduced, clone 2 induced, control (His-FluA-SplitLi-Fok_i + His-Dig-SplitLi-Fok_a) uninduced, control induced)
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In the lanes of the induced clones ( lanes 3,4) a band of about 50 kDA appears which could represent the Fok_a_Venus protein.<br><br>
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A western blot with specific anti-GFP antibodies (first antibody: Santa Cruz Biotechnology sc-9996 mouse αGFP; second antibody: Santa Cruz Biotechnology sc-2005 goat αmouse IgG-HRP) was conducted to prove expression of the complete Fok_a_YFP construct although many Fok_a constructs were hard to express.<br><br>
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[[Image:Freiburg09 WBfokvenus.jpg]]<br>
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Western Blot; pEx Venus-Fok_a in BL21de3; lanes: Marker (NEB prestained protein marker broad range, clone 1 uninduced, clone 1 induced, clone 1 induced, clone 2 uninduced, clone 2 induced, control (His-FluA-SplitLi-Fok_i + His-DigA-SplitLi-Fok_a) uninduced, control induced)
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<br>
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All of the induced clones show a strong signal but also many different bands on the membrane. There is a signal corresponding to 49 kDa which matches the theoretical size of the Fok_a_Venus fusion protein. The bands indicating proteins of smaller size are assumed to be degradation products.
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<br><br>
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===Purification===
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It is possible to use the GFP tag or as in our case YFP for purification by a GFP specific column.<br>
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Due to the lack of time at the end, the purification of the Fok_a proteins by a GFP column is under progress.
 
    
 
    
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'>'''Sequence and Features'''</span>
 
<partinfo>BBa_K243032 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K243032 SequenceAndFeatures</partinfo>
  

Latest revision as of 04:07, 22 October 2009

Fok_a-Venus


This part is composed of the essential Fok_a part protein domain Fok_a of our universal endonuclease, but additionally linked to a Venus-marker to detect even low levels of protein expression by fluorescence microscopy.

Usage and Biology

The addition of a fluorescent protein (Venus) to the protein construct enables easy detection of Fok_a expression by fluorescence microscopy as well as a new way of purification by a GFP-trap column. The Venus protein was C-terminally fused to the Fok_a protein domain. In the opposite orientation, skipped expression of Fok_a would not be detectable. When the fusion protein is expressed by E. coli, the fluorescence signal of the Venus protein can be detected. In addition to these methods, anti-GFP antibodies can be used to check both the protein size and presence by western blotting.

Detection

A strong fluorescence signal could be detected using the fluorescence microscope and IPTG-induced cells. E. coli(RV308) were excited by light of the wavelength 505 nm and fluorescence pictures were made using a cutoff filter.


FokA Venus.JPG

The cell extracts were checked by SDS-page to get information about the expression of proteins.

SDSfokvenus.jpg

SDS gel; pEx Venus-Fok_a in BL21de3; lanes: Marker (ColorPlus Prestained Protein Marker, Broad Range), clone 1 uninduced, clone 1 induced, clone 1 induced, clone 2 uninduced, clone 2 induced, control (His-FluA-SplitLi-Fok_i + His-Dig-SplitLi-Fok_a) uninduced, control induced)

In the lanes of the induced clones ( lanes 3,4) a band of about 50 kDA appears which could represent the Fok_a_Venus protein.

A western blot with specific anti-GFP antibodies (first antibody: Santa Cruz Biotechnology sc-9996 mouse αGFP; second antibody: Santa Cruz Biotechnology sc-2005 goat αmouse IgG-HRP) was conducted to prove expression of the complete Fok_a_YFP construct although many Fok_a constructs were hard to express.

Freiburg09 WBfokvenus.jpg

Western Blot; pEx Venus-Fok_a in BL21de3; lanes: Marker (NEB prestained protein marker broad range, clone 1 uninduced, clone 1 induced, clone 1 induced, clone 2 uninduced, clone 2 induced, control (His-FluA-SplitLi-Fok_i + His-DigA-SplitLi-Fok_a) uninduced, control induced)


All of the induced clones show a strong signal but also many different bands on the membrane. There is a signal corresponding to 49 kDa which matches the theoretical size of the Fok_a_Venus fusion protein. The bands indicating proteins of smaller size are assumed to be degradation products.

Purification

It is possible to use the GFP tag or as in our case YFP for purification by a GFP specific column.
Due to the lack of time at the end, the purification of the Fok_a proteins by a GFP column is under progress.


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 487