Revision as of 03:42, 10 October 2018

sfGFP(BbsI free)

It is a superfolder GFP, a robustly folded version of GFP. It is BbsI restriction site free by mutating two bases from superfolder GFP(BBa_I746916)-from 5’-gaagac-3’ to 5’-gaggat-3’, which does not change the amino acid encoded by this sequence.

Usage and Biology

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.

However, the superfolder GFP (BBa_I746916) contains a BbsI restriction enzyme cleavage site, and the BbsI restriction endonuclease is an economical and efficient enzyme used in goldengate assembly, so the superfolder GFP (BBa_I746916) cannot be used for goldengate assembly. This year we made two point mutations in superfolder GFP (BBa_I746916) to eliminate its BbsI restriction site without changing its amino acid sequence to make it suitable for goldengate assembly. We made it from 5’-gaagac-3’ to 5’-gaggat-3’. We called the mutated superfolder GFP is superfolder GFP mut2 (BBa_K2541401).

Characterization

The improved superfolder GFP mut2 (K2541401) 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 superfolder GFP mut2 (K2541401) was contributed to the Registry of Standard Biological Parts and it will be more widely used in the synthetic biology community.

Sequence and Features