Reporter

Part:BBa_K863113

Designed by: Moritz Müller   Group: iGEM12_Bielefeld-Germany   (2012-09-20)

Cellulose binding Domain of C. cellulovorans cellulose binding protein with Reporter GFP

Figure 1: Predicted structure of the CBM_2-family made with Cn3D

Cellulose binding Domain (CBDclos) of Clostridium cellulovorans cellulose binding protein gene (cbp A) in Standard 25 (Freiburg) under the control of a T7-promoter. This construct was designed as a reporter-fusion-protein to measure the binding capacity of the cellulose binding domain. A GFP is introduced to the C-terminal end of the CBD and connected by a short linker, consisting of four amino acids (Glycine, Serine, Threonine, Glycine).

To that point expression of the protein could not be proven.

Usage and Biology

The CBDclos is an N-terminal domain quite close to the start of the protein-sequence (Figure 2). The NCBI-BLAST identified 92 amino acids (276 bases; green bar) as the protein domain. It belongs to the Cellulose Binding Module family 3 (pfam00942/cl03026; Figure 1) and is part of the very large cellulose binding protein. In which four other carbohydrate binding modules can be found, separated by hydrophobic docking interfaces for cellulase-proteins. This makes the conserved linking sequence unattractive as a Linker for fusion-proteins. For the coding sequence 6 bases (2 amino acids) more than the BLAST predicted were taken as conserved sequence upstream and 6 bases (2AS) downstream of the domain to secure natural folding of the domain.

Figure 2: protein-Blast of the Clostridium cellulovorans cellulose binding protein gene (cbp A)

The expressed GFP-fusion-protein would have 346 amino acids with a molecular weight of 38.8 and a theoretic pI of 5.83. If the protein concentration is measured by optical density at 280 nm the extinction coefficients would be 37945 M-1 cm-1, assuming all pairs of Cys residues form cystines and 37820 M-1 cm-1, assuming all Cys residues are reduced. The ExPASy Prot-Parameter-tool predicted the estimated half-life 30 hours in mammalian reticulocytes, (in vitro) more than 20 hours in yeast (in vivo) and more than 10 hours in Escherichia coli (in vivo). It classified the protein as stable.

The GFP used is (mut3b) [note that this part does not have a barcode] His-Tagged version of gfp from Repressilator reporter. See the design page for more source information.

Fluorescence wavelengths

Cormack et al.Cormack report the following excitation and emission data for GFPmut3 -

  • Excitation max - 501nm
  • Emission max - 511nm

Latency

Cormack et al.Cormack report detectable fluorescence within 8 mins.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1067
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 978


[edit]
Categories
//binding/cellulose
//function/reporter/fluorescence
//primer/reporter/gfp
//primer/t7
//proteindomain/binding
Parameters
emission511
excitation501
familyCellulose Binding Modul family 3 (pfam00942/cl03026)
functioncellulose binding
originClostridium cellulovorans cellulose binding protein gene (cbp A)
proteinGFPmut3b
strain743B
swissproM73817
tagHis6
typeCellulose binding domain
uniprotP38058