Difference between revisions of "Part:BBa K243017:Design"
(→‎Design Notes)
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[https://static.igem.org/mediawiki/parts/0/04/Strepdigasplitfoka.txt Commented GenBank file]
 
[https://static.igem.org/mediawiki/parts/0/04/Strepdigasplitfoka.txt Commented GenBank file]
 
The composite part was cloned step by step. We started with the His-Tag and fused it to DigA. In the next step we fused the SplitLinker behind the DigA, in the final step we fused Fok_i to the construct. All cloning steps to fuse the single parts were accomplished according to the [https://parts.igem.org/Assembly_standard_25 RFC 25].
 
The composite part was cloned step by step. We started with the His-Tag and fused it to DigA. In the next step we fused the SplitLinker behind the DigA, in the final step we fused Fok_i to the construct. All cloning steps to fuse the single parts were accomplished according to the [https://parts.igem.org/Assembly_standard_25 RFC 25].
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We applied the Streptavidine-tag to enable a simultaneous purification of constructs with a His-tag. Strep-tag shows also a higher affinity towards Strep-Tactin than His-tag. For that the purification with Strep-tag is more specific. The used DigA-tag allows the coupling to an Digoxigenin linked oligo. Emanating from our 3D modeling this combination of DigA tagged oligo and the construct containing the protein domain Fok_a is more efficiently than the use of a combination of FluA tagged oligo with a construct containing Fok_a. To avoid interactions between the DigA-tag with the connected protein domain Fok_a we applied the split Linker. This Linker is an improved part of the team Freiburg08 and it is a suitable linker for fusion proteins. The properties of the split linker are an good compromise between the stability and the distance for the connection between protein domain Fok_a and the anticalin tag.
  
 
===Source===
 
===Source===

Revision as of 01:38, 22 October 2009

Strep-DigA-Split Linker-Fok_a


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 278
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1132


Design Notes

The choice of the linker, lipocalin tag and purification tag allows us several different combinations. Commented GenBank file The composite part was cloned step by step. We started with the His-Tag and fused it to DigA. In the next step we fused the SplitLinker behind the DigA, in the final step we fused Fok_i to the construct. All cloning steps to fuse the single parts were accomplished according to the RFC 25.

We applied the Streptavidine-tag to enable a simultaneous purification of constructs with a His-tag. Strep-tag shows also a higher affinity towards Strep-Tactin than His-tag. For that the purification with Strep-tag is more specific. The used DigA-tag allows the coupling to an Digoxigenin linked oligo. Emanating from our 3D modeling this combination of DigA tagged oligo and the construct containing the protein domain Fok_a is more efficiently than the use of a combination of FluA tagged oligo with a construct containing Fok_a. To avoid interactions between the DigA-tag with the connected protein domain Fok_a we applied the split Linker. This Linker is an improved part of the team Freiburg08 and it is a suitable linker for fusion proteins. The properties of the split linker are an good compromise between the stability and the distance for the connection between protein domain Fok_a and the anticalin tag.

Source

Combined the parts by serial cloning steps.

References