Designed by: Memduha Muratoglu   Group: iGEM18_Marburg   (2018-10-02)

Flp recombinase This part encodes the sequence for the Flp recombinase derived from the yeast Saccharomyces cerevisiae (Sadowski 1995). The FLP Recombinase recognizes FRT sites and promotes efficient recombination between two identical FRT recombination sites (Broach et al., 1982). Using the FLP/FRT system genomic engineering in different organisms can be done (Buchholz et al., 1998). The system is similar to the Cre/Lox system. The FRT site consists of palindromic repeats separated by an 8 bp asymmetric core 5'GAAGTTCCTATTCtctagaaaGtATAGGAACTTC3'. Recombination between these sites can lead to insertion, excision, inversion, and translocation of DNA. In our project we used the FLP/FRT system successfully for genomic modifications in V. natriegens.

FLP Recombinase

Sequence and Features

Assembly Compatibility:
  • 10
    Illegal EcoRI site found at 747
    Illegal SpeI site found at 1065
  • 12
    Illegal EcoRI site found at 747
    Illegal SpeI site found at 1065
  • 21
    Illegal EcoRI site found at 747
  • 23
    Illegal EcoRI site found at 747
    Illegal SpeI site found at 1065
  • 25
    Illegal EcoRI site found at 747
    Illegal SpeI site found at 1065
  • 1000
    Illegal EcoRI site found at 747
    Illegal SpeI site found at 1065

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)