Tag

Part:BBa_K2560117

Designed by: Tobias Hensel   Group: iGEM18_Marburg   (2018-09-22)


Phytobrick version of 5a BBa_I11012 Degradation tag

This is the Phytobrick version of the tag I11012 Degradation 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).

Characterization

Our toolbox contains the three degradation tags K2560118 (M0050 5a), K2560119 (M0051 5a) and K2560117 (I11012 5a). Similar to our RBS experiments, we could not use the lux operon as a reporter because the degradation tag is only added to the C-terminal end of the last enzyme encoded in the operon. Therefore, we again used sfGFP as reporter and test constructs were designed with one of the tags fused to the CDS of sfGFP. A non-tagged sfGFP construct serves as the reference.


Figure 1: Fluorescence signal for degradation tag test constructs
The respective degradation tags were appended to the C-terminus of sfGFP. The error bars indicate the standard deviation of four technical replicates.




As expected, appending a degradation tag to a protein decreases its concentration. The strongest decrease and therefore the highest degradation was shown for K2560118 (M0050 5a) with a signal that is not distinguishable from the non-sfGFP expressing control. The second strongest signal reduction was shown for K2560117 (I11012 5a), followed by K2560119 (M0051 5a), which showed the least efficient degradation (figure 1). Our results are in qualitative accordance with the description of these tags for E. coli, which are provided in the registry.
The tested degradation tags belong to the family of SsrA degradation tags. Naturally, they help to degrade incompletely translated proteins by labeling them for Clp proteases (Farrell et al. 2015). We checked for the presence of ClpP, one of the proteases involved in degrading SsrA tagged proteins in E. coli and found a highly homologous protein in V. natriegens. Therefore we assume that the mechanism of Clp mediated degradation is conserved between both organisms, which is in accordance with our results.




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