Tag

Part:BBa_K2560088

Designed by: Tobias Hensel   Group: iGEM18_Marburg   (2018-09-21)


Phytobrick version of 5a FLAG-Tag

This is the Phytobrick version of the tag 5a FLAG-Tag and was build as a part of the Marburg Collection. Instructions of how to use the Marburg Collection are provided at the bottom of the page.

Overview

Protein tags are peptides genetically fused to the proteins of interest. There are many different kinds of tags developed for for different purposes. Protein purification tags are peptide chains with a high affinity to a certain matrix like the histidine affinity tag binding to a Ni2+ matrix. This and other purification tags are used to separate the tagged protein from cell debris ( Kimple et al.2013). Another kind of tags that can be used in a similar way are epitope tags. Here a certain epitope is fused to the protein to be detected by the corresponding antibody. This technique can be used to purify the proteins or to label them in vivo with fluorescent labeled antibodies ( Brizzard B.,2008). Nevertheless for labeling experiments normally the proteins of interest are tagged directly by fluorescent tags. This method can be used to study protein localization, interactions and dynamics ( Crivat G, Taraska JW. ,2012). Degradation tags are another group of tags used to label the proteins in a well recognizable way for proteases reducing their half-life significantly. This makes this tags interesting for synthetic biology enabling the experimenter to influence protein abundances, degradation rates and consequently the cell’s respond ( Cameron DE, Collins J. ,2014).


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]


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)
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Categories
Parameters
None