Part:BBa_K2740015

CR1 nifK

CR1 nifK encodes the subunit beta (NifK) of the molybdenum-iron protein. Together with NifD, they form a α2β2 heterotetramer.

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 856
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1106
  Illegal SapI.rc site found at 1489
  

Parameter of Protein

Number of amino acids: 509

Molecular weight: 56395.40

Theoretical pI: 5.74

Amino acid composition:
Ala (A)  48    9.4%
Arg (R)  26    5.1%
Asn (N)  16   3.1%
Asp (D)  27   5.3%
Cys (C)   9    1.8%
Gln (Q)  19    3.7%
Glu (E)  35    6.9%
Gly (G)  40    7.9%
His (H)  17    3.3%
Ile (I)   23     4.5%
Leu (L)  51  10.0%
Lys (K)  23    4.5%
Met (M)  19   3.7%
Phe (F)  24    4.7%
Pro (P)  26     5.1%
Ser (S)  29     5.7%
Thr (T)  28    5.5%
Trp (W)  2     0.4%
Tyr (Y)  19    3.7%
Val (V)  28    5.5%
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): 62
Total number of positively charged residues (Arg + Lys): 49

Atomic composition:

Carbon      C          2506
Hydrogen    H         3909
Nitrogen    N            681
Oxygen      O          745
Sulfur      S              28

Formula: C2506H3909N681O745S28
Total number of atoms: 7869

Extinction coefficients:

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

Ext. coefficient    39810
Abs 0.1% (=1 g/l)   0.706, assuming all pairs of Cys residues form cystines

 

Ext. coefficient    39310
Abs 0.1% (=1 g/l)   0.697, 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 43.04
This classifies the protein as unstable.

 

Aliphatic index: 82.08

Grand average of hydropathicity (GRAVY): -0.197

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.

Molecular modeling of nifK

To learn more about the molecular structure of ubunit beta (NifK) of the molybdenum-iron protein encoded by nifK, we use Swiss-Model to get the molecular model.

Confirmation of Expression of nifK

We test expression profiles of each structure gene in the nif cluster that overexpressed in EJNC by conducting Real-time Quantitative PCR(qPCR). Relative expression compared to the housekeeping gene 16S rRNA is shown.

qRT-PCR analysis demonstrates that all the component genes of the nif cluster are significantly over expressed in EJNC whereas the transcription of these genes are no detected (N.D.) in nondiazotrophic E.coli JM109. Based on these analysis, we know nifK has a relatively low expression level.

Usage

In our this year’s project, we intends to establish a sound and ideal whole-cell photocatalytic nitrogen fixation system. We use the engineered E. coli cells to express nitrogenase and in-situ synthesize of CdS semiconductors in the biohybrid system. Instead of ATP-hydrolysis, such system is able to photocatalytic N2(nitrogen) to NH3(ammonia). The biohybrid system based on engineered E. coli cells with biosynthesis inorganic materials will likely become an alternative approach for the convenient utilization of solar energy. So, certainly we need not only a powerful solar power transition system but also a strong nitrogen fixation system to improve the efficiency of our whole-cell photocatalytic nitrogen fixation system. According to the above requirements, we choose a different nif gene cluster from Paenibacillus polymyxa CR1 to test its expression level.