Coding

Part:BBa_K4390064

Designed by: Maarten van den Ancker   Group: iGEM22_Edinburgh-UHAS_Ghana   (2022-09-03)
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SpyTagged sfGFP

This part is not compatible with BioBrick RFC10 assembly but is compatible with the iGEM Type IIS Part standard which is also accepted by iGEM.

This is a Level 0 part of type O-C part generating the following 4 base overhangs at upstream (AGCC) and downstream (GCTT) ends.

Usage and Biology

SpyTag is a 13 amino acid-long protein that spontaneously reacts with the SpyCatcher protein (12.3 kDa). Together they form an intermolecular isopeptide bond. This provides an alternative to His-tag purification techniques. Insertion of either the SpyTag or the SpyCatcher sequence into a recombinant protein will allow its purification.

The green fluorescent protein (GFP) is a protein that can produce bright green colour. Due to these properties, it has always been used as reporter of protein expression. Protein fused with sfGFP will turn the colony green if expressed, which can be used to identify the ability of certain genes to be expressed in other organism like E. coli. Superfolder GFP (sfGFP) is a more robustly folded version of GFP, which is derived from GFP that often misfolds when expressed as fusions with other proteins. Superfolder GPF shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics.

This part contains Spytagged sfGFP which is used to test whether the Spytag-Spycatcher hydrogel will form correctly with desired protein inside. If the Spytag-Spycatcher forms, the gel will present bright green colour due to presence of GFP.

Design

This is a type OC part, so it combines the 5' fusion site of a type O part with the 3' fusion site of a type C part. This was done so that this construct fits in the expression cassette with the rest of our modular proteins, as the TU for this protein part has fewer parts than the others.

The 5' fusion site starts with GCC encoding alanine so that the SpyTag starts in line, this works with the type N parts we have designed. Since it is the last protein part in the ORF, we end with a double stop and then the standard JUMP type C 3' fusion site.

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]


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Categories
Parameters
None