Coding

Part:BBa_K2560270

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


SXT operon

This part encodes the SXT operon consisting of the phage proteins: SXT-Exo, SXT-Beta and lambda-Gam, under the control of an arabinose promoter. This system can be used for recombineering in V. natriegens. It is similar to the lambda-Gam system used for recombineering in E. coli. SXT-Exo is a nuclease, digesting linear DNA from the 5' end and creates 3' overhangs. These 3' overhangs are protected from degradation by SXT-Beta and the lambda-Gam protein prevents endogenous nucleases from degrading the linear DNA introduced into the V. natriegens cells. This way, the SXT-system improves the homologous recombination event.


Recombineering with the SXT-system

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 496
    Illegal EcoRI site found at 2005
    Illegal PstI site found at 754
    Illegal PstI site found at 1001
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 496
    Illegal EcoRI site found at 2005
    Illegal PstI site found at 754
    Illegal PstI site found at 1001
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 496
    Illegal EcoRI site found at 2005
    Illegal BamHI site found at 2066
    Illegal XhoI site found at 3318
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 496
    Illegal EcoRI site found at 2005
    Illegal PstI site found at 754
    Illegal PstI site found at 1001
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 496
    Illegal EcoRI site found at 2005
    Illegal PstI site found at 754
    Illegal PstI site found at 1001
    Illegal AgeI site found at 574
    Illegal AgeI site found at 2231
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2248


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