Part:BBa_K2541400

sfGFP_optimism

1. Usage and Biology

Green fluorescent protein (GFP) 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 (sfGFP), was developed and described by Pédelacq et al at 2006[3] that folds well even when fused to poorly folded polypeptides. We decided to use sfGFP as our reporter protein due to its faster folded feature and better fluorescence intensity. The superfolder GFP had been registered in iGEM BBa_I746916. There is another superfolder GFP designed by Overkamp W et al at 2013[4], which is a codon optimized sfGFP. 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_optimism). Compared to superfolder GFP (BBa_I746916), sfGFP_optimism (BBa_K2541400) is BbsI restriction site free, and the BbsI restriction endonuclease is an economical and efficient enzyme used in GoldenGate assembly, so sfGFP_optimism can be used in GoldenGate assembly to achieve efficient and rapid assembly of gene fragments.

2. Characterization

The free of the BbsI recognition site was confirmed by nucleic acid electrophoresis (figure 1).

Figure 1.

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Then, we got the emission and excitation spectra of two type sfGFP: sfGFP_optimism (BBa_K2541400) and superfolder GFP (BBa_I746916) (figure 2).

sfGFP_optimism f2

Figure 2.

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For further characterization, we detected the expression intensity of these two types of sfGFP. According to the results (figure 3), we found out that the fluorescence intensity of sfGFP_optimism (BBa_K2541400) is nearly 2.6 as much as superfolder GFP (BBa_I746916).

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]