Difference between revisions of "Part:BBa K2255003"

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====GFP purification with a Ni column====
 
====GFP purification with a Ni column====
  
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[[File:T--Aix-Marseille--gelMtag.png|150px|right|thumb|A)Loading sample. B)Wash. C)Elution.]]
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[[File:T--Aix-Marseille--gelMtag.png|200px|right|thumb|A)Loading sample. B)Unbound. C)Elution.]]
  
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Purification of [[https://parts.igem.org/Part:BBa_K1321337 sfGFP]] fusionned with this tag (BBa_K2255003), with the [[https://www.qiagen.com/fr/shop/sample-technologies/protein/expression-purification-detection/ni-nta-spin-kit/#orderinginformation Ni-NTA Spin Kit]] (Quiagen). As you can see, mainly sfGFP is present after elution and most of the protein weren't bound. Thus, the his-tag of this part is functionnal.
  
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Purification of [[https://parts.igem.org/Part:BBa_K1321337 sfGFP]] fusionned with this tag (BBa_K2255003), with the [[https://www.qiagen.com/fr/shop/sample-technologies/protein/expression-purification-detection/ni-nta-spin-kit/#orderinginformation Ni-NTA Spin Kit]] (Quiagen). As you can see, mainly sfGFP is present after elution and most of the protein are gone with the wash. Thus, the his-tag of this part is functionnal.
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In our [http://2017.igem.org/Team:Aix-Marseille/DEPS project], we wanted to purify the [https://parts.igem.org/Part:BBa_K2255006 EPS Depolymerase] for its use in our cure. Thank to this tag, we will be able to successfully purify our protein with a single Ni column and this will enable us to use the [https://parts.igem.org/Part:BBa_K2255006 EPS Depolymerase] as we wanted and test its activity.
  
 
==References==
 
==References==

Latest revision as of 09:34, 24 October 2017


Multi-Tag (Rfc 25)

This tag provides multiple fixation sites: a Strep-tag [1] and a histidine tag. Between those tag is a TEV protease cutting site [2].

This part was designed with the Freiburg (Rfc25) extension. Thus, it contains the restriction sites NgoMIV and AgeI that are compatible and allow the absence of a start and stop codon, which ease the assembly of multiple protein domain.

Usage and Biology

This part is usefull in our to purify the enzyme that use to disrupt the biofilm. Check out our [http://2017.igem.org/Team:Aix-Marseille/DEPS enzyme design page] to know more about our project.

GFP purification with a Ni column

A)Loading sample. B)Unbound. C)Elution.

Purification of [sfGFP] fusionned with this tag (BBa_K2255003), with the [Ni-NTA Spin Kit] (Quiagen). As you can see, mainly sfGFP is present after elution and most of the protein weren't bound. Thus, the his-tag of this part is functionnal.

In our [http://2017.igem.org/Team:Aix-Marseille/DEPS project], we wanted to purify the EPS Depolymerase for its use in our cure. Thank to this tag, we will be able to successfully purify our protein with a single Ni column and this will enable us to use the EPS Depolymerase as we wanted and test its activity.

References

  1. ↑ Schmidt, Thomas GM; Skerra, Arne (2007). "The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins". Nature Protocols. 2 (6): 1528–35. PMID 17571060. doi:10.1038/nprot.2007.209
  2. ↑ Parks TD, Leuther KK, Howard ED, Johnston SA, Dougherty WG (February 1994). "Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase". Anal. Biochem. 216 (2): 413–7. PMID 8179197. doi:10.1006/abio.1994.1060

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]