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

We constrcted sfGFP_optimism (BBa_K2541400) sequence and superfolder GFP (BBa_I746916) on the pSB1C3 vector. The BbsI recognition site free was confirmed by nucleic acid electrophoresis (figure 1). And the length of these sequences are correct.

Figure 1. L1: 1kb DNA marker; L2: BBa_I746916; L3: BBa_I746916+BbsI; L4: BBa_K2541401; L5: BBa_K2541401+BbsI; L6: BBa_K2541400; L7: BBa_K2541401+BbsI; L8: 1kb DNA marker. The coding sequence of sfGFP_Bbs I free and sfGFP_optimism can not be digested by Bbs I.

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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 times higher than superfolder GFP (BBa_I746916).

Figure 3.

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We have made an improvement on the superfolder GFP (BBa_I746916). Our sfGFP_optimism (BBa_K2541400) is BbsI restriction site free, which can be used in GoldenGate assembly to achieve efficient and rapid assembly of gene fragments. And its fluorescence intensity is higher than superfolder GFP (BBa_I746916).

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]