Difference between revisions of "Part:BBa K2541400"
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<h1>'''Biology and Usage'''</h1>
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<h1>'''1. Usage and Biology'''</h1>
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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].  
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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].  
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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. 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 codon optimized sfGFP. It was be used in ''Escherichia coli'' by Segall-Shapiro T H et al at 2018[5].
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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.
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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].
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<h1>'''2. Characterization'''</h1>
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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).
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<h1>'''Characterization'''</h1>
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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 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.  
 
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.  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2541400 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2541400 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2541400 parameters</partinfo>
 
<partinfo>BBa_K2541400 parameters</partinfo>
 
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Revision as of 10:03, 14 October 2018


sfGFP_optimism

It is a superfolder GFP, a robustly folded version of GFP, which is BbsI restriction site free and can be used in GoldenGate assembly.

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, was developed and described by Pédelacq et al at 2006 [3]that folds well even when fused to poorly folded polypeptides. 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 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 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]