Composite

Part:BBa_K2541403

Designed by: Shuting Zheng   Group: iGEM18_Jilin_China   (2018-10-05)
Revision as of 13:14, 10 October 2018 by Shuting (Talk | contribs)


superfolder GFP measurement device

It is a measurement device for superfolder GFP (BBa_I746916) fluorescence intensity, which is composed of promoter (BBa_J23104), RBS (BBa_B0034), superfolder GFP (BBa_I746916) and terminator (BBa_B0015).

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.

So, this year our team improved the superfolder GFP (BBa_I746916). Firstly, we registered the superfolder GFP designed by Overkamp W et al[4] 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. Secondly, 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 sfGFP(BbsI free) (BBa_K2541401).

In order to compare the two sfGFP we designed with superfolder GFP (BBa_I746916), we designed this measurement device to measure superfolder GFP (BBa_I746916) fluorescence intensity.


Characterization



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 74


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Parameters
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