Revision as of 17:29, 15 October 2018

superfolder GFP coding sequence

This is the coding sequence of superfolder GFP (Pedelacq et al (2006): "Engineering and characterization of a superfolder green fluorescent protein", Nature Biotech 24 (1) January 2006).

More information about the properties of this GFP in relation to the currently used mut3GFP can be found at: http://openwetware.org/wiki/IGEM:Cambridge/2008/Improved_GFP.

It carries the following amino acid changes with respect to mut3 GFP (E0040), the currently most commonly used GFP in the registry:

S30R, Y39N, F64L, G65T, F99S, N105T, Y145F, M153T, V163A, I171V, A206V

Its in-vivo properties are considerably improved with respect to mut3 - it develops fluorescence about 3fold faster than mut3 GFP and reaches 4fold higher absolute fluorescence levels. Fluorescenct colonies can be identified with the naked eye even without UV or blue light illumination (that is to say the amount of blue light in normal daylight or lablight is sufficient). Additionally it is more stable in vitro and refolds faster after in vitro denaturation with respect to mut3 GFP.

Note: Superfolder GFP is available in constructs driven by the pBAD and T7 promoters: part numbers I746908 and I746909 respectively. Additionally 6-his tagged versions for protein purification exist: I746914 (pBAD driven) and I746915 (T7 driven).

Contribution

Group: Valencia_UPV iGEM 2018
Author: Adrián Requena Gutiérrez
Summary: We have adapted the part to be able to assemble transcriptional units with the Golden Gate method and we have done the characterization of this protein.
Documentation:
We adapted the CDS BBa_I746916 to be used to assemble composite parts using the Golden Gate method creating BBa_K2656013. Then we made the characterization of the part: The characterization of this protein (and by extension of all the other part that codify for the sfGFP) was performed with our transcriptional unit BBa_K2656101. This transcriptional unit was assembled in a [http://2018.igem.org/Team:Valencia_UPV/Design Golden Braid alpha 1 plasmid] including the following parts:

• BBa_K2656004: the J23106 promoter in its Golden Braid compatible version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]
• BBa_K2656009: the B0030 ribosome biding site in its Golden Braid compatible version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]
• BBa_K2656013: This part.
• BBa_K2656026: the B0015 transcriptional terminator in its Golden Braid compatible version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]

In order to carry out a correct characterization of the protein and to be able to use it to make measurements of the different transcriptional units that we have assembled with it, we have obtained the emission and excitation spectra in the conditions of our equipment. By using this [http://2018.igem.org/Team:Valencia_UPV/Experiments#spectra protocol] with the parameters of Table 1, Figure 1 has been obtained.

Figure 1. sfGFP emission and excitation spectra

Improvement

Group: iGEM2018_Jilin_China
Author:Zihao Wang
Summary: 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.
Document:

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].

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 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 13