Part:BBa_K2740012

CR1 nifB

CR1 nifB encodes nitrogen fixation protein NifB that is essential for biosynthesis of the active-site nitrogenase cofactor. If the CR1 nifB was deleted, the nitrogen fixation would not happen.

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
  COMPATIBLE WITH RFC[1000]

Parameter of Protein

 

Number of amino acids: 499

Molecular weight: 54885.93

Theoretical pI: 7.24

Amino acid composition:
Ala (A)  45    9.0%
Arg (R)  35    7.0%
Asn (N)  18   3.6%
Asp (D)  23   4.6%
Cys (C)  16    3.2%
Gln (Q)  19    3.8%
Glu (E)  38    7.6%
Gly (G)  42    8.4%
His (H)  17    3.4%
Ile (I)    29   5.8%
Leu (L)  41    8.2%
Lys (K)  26    5.2%
Met (M)  12   2.4%
Phe (F)  13    2.6%
Pro (P)  25     5.0%
Ser (S)  28     5.6%
Thr (T)  16    3.2%
Trp (W)  2     0.4%
Tyr (Y)  13    2.6%
Val (V)  41    8.2%
Pyl (O)   0     0.0%
Sec (U)   0    0.0%

 (B)   0         0.0%
(Z)   0   0.0%
(X)   0         0.0%

 

Total number of negatively charged residues (Asp + Glu): 61
Total number of positively charged residues (Arg + Lys): 61

Atomic composition:

Carbon      C          2398
Hydrogen    H         3853
Nitrogen    N            703
Oxygen      O          716
Sulfur      S              28

Formula: C2398H3853N703O716S28
Total number of atoms: 7698

Extinction coefficients:

Extinction coefficients are in units of  M-1 cm-1, at 280 nm measured in water.

Ext. coefficient    31370
Abs 0.1% (=1 g/l)   0.572, assuming all pairs of Cys residues form cystines

 

Ext. coefficient    30370
Abs 0.1% (=1 g/l)   0.553, assuming all Cys residues are reduced

Estimated half-life:

The N-terminal of the sequence considered is M (Met).

The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro).
>20 hours (yeast, in vivo).
>10 hours (Escherichia coli, in vivo).

 

Instability index:

The instability index (II) is computed to be 43.00
This classifies the protein as unstable.

 

Aliphatic index: 87.56

Grand average of hydropathicity (GRAVY): -0.254

Design Notes

Nitrogenase is a complex enzyme system consisting of nine protein components.Additionally, to maintain stoichiometry of these protein components is an essential requirement for nitrogenase biosynthesis and activity. However, there is only one copy of each structure gene present in the nif gene cluster. Therefore, cloning each of these nif genes and setting as independent part can facilitate the regulation of balancing expression ratios from the transcription and/or translation level(s) when they are heterogeneously expressed in non-diazotrophic hosts.We sent the sequences of the PCR template to synthesis, but unfortunately, EcoRI and PstI striction enzyme cut site was involved after they promoted it again. But the part can be manipulated by XbaI and SpeI or can be assembled by gibson assembly,that is what we did.