Composite
CBDclos(T7

Part:BBa_K863112

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

Cellulose binding domain of C. cellulovorans with T7, RBS, GS-Linker (Freiburg-Standard)

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 a control of a T7-promoter. This construct was designed to attach proteins of interest via a Standard 25 assembly (Freiburg) to the C-terminal end of the CBD which carries a short linker.

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 protein would have 100 amino acids with a molecular weight of 10.9 and a theoretic pI of 4.56. If the protein concentration is measured by optical density at 280 nm the extinction coefficients would be 15930 M-1 cm-1, assuming all pairs of Cys residues form cystines and 15930 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. 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 329
  • 1000
    COMPATIBLE WITH RFC[1000]


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