Difference between revisions of "Part:BBa K1483003:Experience"

 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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===Applications of BBa_K1483003===
 
===Applications of BBa_K1483003===
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== Contribution by Team Tuebingen 2015: ==
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We expressed the ssp GyrB split intein as a fusion protein with an enzyme weighing 52.2 kDa (BBa_K1483000) using the pETue vector (BBa_K1680026) in BL21(DE3).  We were able to show that the protein can be expressed and does react with the synthetic peptide described in the design section. We were able to confirm this using a bandshift assay, which uses the intein's protein-splicing mechanism.
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'''Expression:'''
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This part can be expressed by growing a culture transformed with the above mentioned plasmid to OD(600)=0.3, inducing with 2 mM IPTG and expressing over night at room temperature. The culture can then be lysed and the protein purified using the 6xHis-Tag. The result of such an experiment is shown below. The band at 60 kDa in the eluate fraction represents the NAGA-Intein fusion protein.
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<p>
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https://static.igem.org/mediawiki/2015/6/69/Team_Tuebingen_Gel_InteinNaga_Purification.png
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</p>
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To establish that the split intein interacts and is active in comination with the synthetic peptide described in the design section, we incubated the peptide and the purified protein for 48 hours at room temperature. In the gel shown below, after addition of the peptide, an additional band appears slighly below 60 kDa.
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<p>
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https://static.igem.org/mediawiki/2015/2/24/Team_Tuebingen_Gel_InteinNaga_Peptide_2.png
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</p>
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The NAGA-Intein construct has either reacted in the predicted way, or a side reaction called N-Cleavage has occured. During N-Cleavage, the the N-Extein is cleaved from the intein without a splicing reaction taking place. For a discussion on this and further experiments to establish if the expected reacion, or the N-Cleavage takes place see our WIKI!
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We have slightly improved the graphic on the peptide. It is important to know where exactly the intein ends and the extein begins. To this end, we have color coded the extein region red. Very simply this means that the Intein from the first aminoacids following what is coded by the RFC suffix is excised from the fusion protein and replaced what with the red part of the peptide shown below.
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<p>
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https://static.igem.org/mediawiki/2015/2/22/Team_Tuebingen_15_Synth-Intein_Tue15.png
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</p>
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'''Correct buffer:'''
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The correct buffer conditions in which to use this intein can be found in "Novel Split Intein for trans-Splicing Synthetic Peptide onto C-Terminus of Protein" by Liu et al. (2009). They suggest using ten times the molar amount of peptide compared to the amount of protein. The optimal buffer contitions are 20 mM Tris-HCl at pH = 8.0, 150 mM NaCl and 1 mM EDTA.
  
 
===User Reviews===
 
===User Reviews===

Revision as of 02:41, 19 September 2015

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1483003

Contribution by Team Tuebingen 2015:

We expressed the ssp GyrB split intein as a fusion protein with an enzyme weighing 52.2 kDa (BBa_K1483000) using the pETue vector (BBa_K1680026) in BL21(DE3). We were able to show that the protein can be expressed and does react with the synthetic peptide described in the design section. We were able to confirm this using a bandshift assay, which uses the intein's protein-splicing mechanism.

Expression: This part can be expressed by growing a culture transformed with the above mentioned plasmid to OD(600)=0.3, inducing with 2 mM IPTG and expressing over night at room temperature. The culture can then be lysed and the protein purified using the 6xHis-Tag. The result of such an experiment is shown below. The band at 60 kDa in the eluate fraction represents the NAGA-Intein fusion protein.

Team_Tuebingen_Gel_InteinNaga_Purification.png

To establish that the split intein interacts and is active in comination with the synthetic peptide described in the design section, we incubated the peptide and the purified protein for 48 hours at room temperature. In the gel shown below, after addition of the peptide, an additional band appears slighly below 60 kDa.

Team_Tuebingen_Gel_InteinNaga_Peptide_2.png

The NAGA-Intein construct has either reacted in the predicted way, or a side reaction called N-Cleavage has occured. During N-Cleavage, the the N-Extein is cleaved from the intein without a splicing reaction taking place. For a discussion on this and further experiments to establish if the expected reacion, or the N-Cleavage takes place see our WIKI!

We have slightly improved the graphic on the peptide. It is important to know where exactly the intein ends and the extein begins. To this end, we have color coded the extein region red. Very simply this means that the Intein from the first aminoacids following what is coded by the RFC suffix is excised from the fusion protein and replaced what with the red part of the peptide shown below.


Team_Tuebingen_15_Synth-Intein_Tue15.png

Correct buffer: The correct buffer conditions in which to use this intein can be found in "Novel Split Intein for trans-Splicing Synthetic Peptide onto C-Terminus of Protein" by Liu et al. (2009). They suggest using ten times the molar amount of peptide compared to the amount of protein. The optimal buffer contitions are 20 mM Tris-HCl at pH = 8.0, 150 mM NaCl and 1 mM EDTA.

User Reviews

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