Part:BBa_K2740020

CR1 nifV

CR1 nifV encodes homocitrate synthase NifV that enables the synthesis of the homocitrate moiety of the molybdenum-iron protein cofactor.

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: 378

Molecular weight: 41329.16

Theoretical pI: 5.45

Amino acid composition:
Ala (A)  45  11.9%
Arg (R)  25    6.6%
Asn (N)   9   2.4%
Asp (D)  19   5.0%
Cys (C)   5    1.3%
Gln (Q)  14    3.7%
Glu (E)  31    8.2%
Gly (G)  30    7.9%
His (H)  11    2.9%
Ile (I)   22     5.8%
Leu (L)  40  10.6%
Lys (K)  13    3.4%
Met (M)  10   2.6%
Phe (F)   9     2.4%
Pro (P)  14     3.7%
Ser (S)  24     6.3%
Thr (T)  16    4.2%
Trp (W)  5     1.3%
Tyr (Y)   9    2.4%
Val (V)  27    7.1%
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): 50
Total number of positively charged residues (Arg + Lys): 38

Atomic composition:

Carbon      C          1821
Hydrogen    H         2910
Nitrogen    N            516
Oxygen      O          551
Sulfur      S              15

Formula: C1821H2910N516O551S15
Total number of atoms: 5813

Extinction coefficients:

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

Ext. coefficient    41160
Abs 0.1% (=1 g/l)   0.996, assuming all pairs of Cys residues form cystines

 

Ext. coefficient    40910
Abs 0.1% (=1 g/l)   0.990, 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 43.86
This classifies the protein as unstable.

 

Aliphatic index: 96.59

Grand average of hydropathicity (GRAVY): -0.087

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.