Tag

Part:BBa_K2560257:Design

Designed by: Daniel Marchal   Group: iGEM18_Marburg   (2018-09-29)


4x Streptag for N-terminal protein tagging

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]


Design Notes

To have a small linker to the 3' coding region, a GGG (glycine) was added as well as an ATG (methionine) as startcodon. This region is flanked by overhangs which are Phytobrick- and MoClo-compatible and by two BsaI recognition sites (Weber et al., 2011). It was built with the Marburg Toolbox, a golden gate based toolbox for modular cloning. According to the Marburg Toolbox, the part is designed as a 4X-part (CDS).


If the Streptag shall be fused to the 5' end (N-terminus of the protein), use this part, for 3' fusion (C-terminus of the protein) use BBa_K2560258.

GGTCTCGAATG-coding_region-GATGTGAGACC

The sequence was codonoptimized for V. natriegens ATCC 14048.

Source

Source of the part:

The sequence of this part was used from Addgene (Plasmid #55181). To have a small linker to the 3' coding region, a GGG (glycine) was added as well as an ATG (methionine) as startcodon.

The part was codonoptimized for V. natriegens and then synthetisized by IDT and integrated into the vector BBa_K2560002 via BsmBI

References

Weber E., Engler C., Gruetzner R., Werner S., Marillonnet S. (2011). A modular cloning system for standardized assembly of multigene constructs. PLoS One 6:e16765. 10.1371/journal.pone.0016765

Marburg Toolbox

We proudly present the Marburg Collection, a novel golden-gate-based toolbox containing various parts that are compatible with the PhytoBrick system and MoClo. Compared to other bacterial toolboxes, the Marburg Collection shines with superior flexibility. We overcame the rigid paradigm of plasmid construction - thinking in fixed backbone and insert categories - by achieving complete de novo assembly of plasmids.

36 connectors facilitate flexible cloning of multigene constructs and even allow for the inversion of individual transcription units. Additionally, our connectors function as insulators to avoid undesired crosstalk.

The Marburg Collection contains 123 parts in total, including:
inducible promoters, reporters, fluorescence and epitope tags, oris, resistance cassettes and genome engineering tools. To increase the value of the Marburg Collection, we additionally provide detailed experimental characterization for V. natriegens and a supportive software. We aspire availability of our toolbox for future iGEM teams to empower accelerated progression in their ambitious projects.


Figure 3: Hierarchical cloning is facilitated by subsequent Golden Gate reactions.
Basic building blocks like promoters or terminators are stored in level 0 plasmids. Parts from each category of our collection can be chosen to built level 1 plasmids harboring a single transcription unit. Up to five transcription units can be assembled into a level 2 plasmid.
Figure 4: Additional bases and fusion sites ensure correct spacing and allow tags.
Between some parts, additional base pairs were integrated to ensure correct spacing and to maintain the triplet code. We expanded our toolbox by providing N- and C- terminal tags by creating novel fusions and splitting the CDS and terminator part, respectively.


Parts of the Marburg Toolbox




Tags and Entry Vectors




  • K2560001 (Entry Vector with RFP dropout)
  • K2560002 (Entry Vector with GFP dropout)
  • K2560005 (Resistance Entry Vector with RFP Dropout)
  • K2560006 (Resistance Entry Vector with GFP Dropout)
  • K2560305 (gRNA Entry Vector with GFP Dropout)