Part:BBa_K2740014

CR1 nifD

CR1 nifD encodes the subunit alpha (NifD) of the molybdenum-iron (MoFe) protein.

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: 482

Molecular weight: 54204.38

Theoretical pI: 5.96

Amino acid composition:
Ala (A)  31    6.4%
Arg (R)  25    5.2%
Asn (N)  14   2.9%
Asp (D)  27   5.6%
Cys (C)  11    2.3%
Gln (Q)  15    3.1%
Glu (E)  39    8.1%
Gly (G)  47    9.8%
His (H)  13    2.7%
Ile (I)   41     8.5%
Leu (L)  29    6.0%
Lys (K)  33    6.8%
Met (M)  23   4.8%
Phe (F)  16    3.3%
Pro (P)  20     4.1%
Ser (S)  24     5.0%
Thr (T)  18    3.7%
Trp (W)  7     1.5%
Tyr (Y)  20    4.1%
Val (V)  29    6.0%
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): 66
Total number of positively charged residues (Arg + Lys): 58

Atomic composition:

Carbon      C          2405
Hydrogen    H         3770
Nitrogen    N            652
Oxygen      O          706
Sulfur      S              34

Formula: C2405H3770N652O706S34
Total number of atoms: 7567

Extinction coefficients:

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

Ext. coefficient    68925
Abs 0.1% (=1 g/l)   1.272, assuming all pairs of Cys residues form cystines

 

Ext. coefficient    68300
Abs 0.1% (=1 g/l)   1.260, 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 43.69
This classifies the protein as unstable.

 

Aliphatic index: 80.52

Grand average of hydropathicity (GRAVY): -0.294          

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.