Difference between revisions of "Part:BBa K2740013"

Revision as of 10:59, 15 October 2018

CR1 nifH

CR1 nifH encodes nitrogenase reductase NifH, which is an electron donor to the molybdenum-iron (MoFe) protein, contributing to the electron transport in the nitrogen fixation system.

Sequence and Features

Parameter of Protein

Number of amino acids: 288

Molecular weight: 31494.95

Theoretical pI: 4.78

Amino acid composition:
Ala (A)  26    9.0%
Arg (R)  13    4.5%
Asn (N)  15   5.2%
Asp (D)  14   4.9%
Cys (C)   6    2.1%
Gln (Q)  13    4.5%
Glu (E)  28    9.7%
Gly (G)  28    9.7%
His (H)   4    1.4%
Ile (I)    22   7.6%
Leu (L)  27    9.4%
Lys (K)  14    4.9%
Met (M)  11   3.8%
Phe (F)   8     2.8%
Pro (P)   8     2.8%
Ser (S)   9     3.1%
Thr (T)  17    5.9%
Trp (W)  0     0.0%
Tyr (Y)   8    2.8%
Val (V)   17   5.9%
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): 42
Total number of positively charged residues (Arg + Lys): 27

Atomic composition:

Carbon      C          1372
Hydrogen    H         2213
Nitrogen    N            377
Oxygen      O          435
Sulfur      S              17

Formula: C1372H2213N377O435S17
Total number of atoms: 4414

Extinction coefficients:

This protein does not contain any Trp residues. Experience shows that
this could result in more than 10% error in the computed extinction coefficient.

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

Ext. coefficient    12295
Abs 0.1% (=1 g/l)   0.390, assuming all pairs of Cys residues form cystines

Ext. coefficient    11920
Abs 0.1% (=1 g/l)   0.378, 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 39.05
This classifies the protein as stable.

Aliphatic index: 92.50

Grand average of hydropathicity (GRAVY): -0.161

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 nifH

To learn more about the molecular structure of nitrogenase reductase NifH encoded by nifH, we use Swiss-Model to get the molecular model of nitrogenase reductase NifH.

Confirmation of Expression of nifH

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 nifH has a relatively moderate expression level.

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.