Part:BBa_K2541400

sfGFP_optimism

Biology and Usage

Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo, and many different variants are currently available to study bacterial cell biology. It exhibits intrinsic fluorescence and is commonly used as a reporter gene in intact cells and organisms [1]. Many mutants of the protein with either modified spectral properties, increased fluorescence intensity, or improved folding properties have been reported [2].

GFP often misfold when expressed as fusions with other proteins, while a robustly folded version of GFP, called superfolder GFP, was developed and described by Pédelacq et al at 2006 [3]that folds well even when fused to poorly folded polypeptides. Furthermore, sfGFP might be particularly suitable for gene expression studies, since the emergence of fluorescence closely matches induction of transcription. The superfolder GFP had been registered in iGEM BBa_I746916. Its codon usage is a compromise for optimum expression in E. coli and B. subtilis. There is another superfolder GFP designed by Overkamp W et al at 2013[4], which is codon optimized for S. pneumoniae. It was be used in Escherichia coli by Segall-Shapiro T H et al at 2018[5].

This year our team registered the superfolder GFP designed by Overkamp W et al with a BBa_K2541400 (called sfGFP). Compared to superfolder GFP(BBa_I746916), sfGFP (BBa_K2541400) is BbsI restriction site free, so it can be used in GoldenGate assembly to achieve efficient and rapid assembly of gene fragments. And sfGFP (BBa_K2541400) has x fold increased fluorescence intensity than superfolder GFP(BBa_I746916).

Characterization

The improved sfGFP (K2541400) variant was standardised into BioBrick format and its fluorescence intensity was compared with superfolder GFP(BBa_I746916) .Its x fold increased fluorescence intensity is a very useful feature. And it is BbsI restriction site free, so it can be used in GoldenGate assembly to achieve efficient and rapid assembly of gene fragments.

The new variant sfGFP (K2541400) was contributed to the Registry of Standard Biological Parts and it will be more widely used in the synthetic biology community.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 421
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]