Difference between revisions of "Part:BBa K2740017"
 
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   <h2>Design Notes</h2>
 
   <h2>Design Notes</h2>
 
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<p align="left">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.</p>
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<p align="left">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.</p>

Latest revision as of 10:16, 8 October 2018


CR1 nifN

CR1 nifN encodes the cofactor NifN for the maturation of functional molybdenum-iron 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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 525
  • 1000
    COMPATIBLE WITH RFC[1000]


Parameter of Protein

Number of amino acids: 435

Molecular weight: 46939.70

Theoretical pI: 6.18

Amino acid composition:
Ala (A)  44  10.1%
Arg (R)  24    5.5%
Asn (N)   7   1.6%
Asp (D)  21   4.8%
Cys (C)   4    0.9%
Gln (Q)  13    3.0%
Glu (E)  26    6.0%
Gly (G)  38    8.7%
His (H)  14    3.2%
Ile (I)   20     4.6%
Leu (L)  52  12.0%
Lys (K)  17    3.9%
Met (M)  11   2.5%
Phe (F)  15    3.4%
Pro (P)  22     5.1%
Ser (S)  41     9.4%
Thr (T)  23    5.3%
Trp (W)  6     1.4%
Tyr (Y)   8    1.8%
Val (V)  29    6.7%
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): 47
Total number of positively charged residues (Arg + Lys): 41

Atomic composition:

Carbon      C          2087
Hydrogen    H         3318
Nitrogen    N            578
Oxygen      O          622
Sulfur      S              15

Formula: C2087H3318N578O622S15
Total number of atoms: 6620

Extinction coefficients:

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

Ext. coefficient    45170
Abs 0.1% (=1 g/l)   0.962, assuming all pairs of Cys residues form cystines

 

Ext. coefficient    44920
Abs 0.1% (=1 g/l)   0.957, assuming all Cys residues are reduced

Estimated half-life:

The N-terminal of the sequence considered is L (Leu).

The estimated half-life is: 5.5 hours (mammalian reticulocytes, in vitro).
3 min (yeast, in vivo).
2 min (Escherichia coli, in vivo).

 

Instability index:

The instability index (II) is computed to be 38.18
This classifies the protein as stable.

 

Aliphatic index: 94.00

Grand average of hydropathicity (GRAVY): -0.017

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.