Difference between revisions of "Part:BBa K2740015"

Latest revision as of 13:56, 16 October 2018

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

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

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 engineered E.coli JM109 (EJNC). E.coli JM109 (EJ) severs as control by conducting Real-time Quantitative PCR(qPCR). Relative expression compared to the housekeeping gene 16S rRNA is shown. So we can know the expression level of nifK in the E.coli JM109 (EJ).

Figure 1. Expression profiles of each structure gene in the nif cluster that overexpressed in engineered E.coli JM109 (EJNC). E.coli JM109 (EJ) severs as control and relative expression compared to the housekeeping gene 16S rRNA is shown. N.D. represent not ditected.

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.

Reference

1. Wang, L., et al., A minimal nitrogen fixation gene cluster from Paenibacillus sp. WLY78 enables expression of active nitrogenase in Escherichia coli. PLoS Genet, 2013. 9(10): p. e1003865.
2. Fixen, K.R., et al., Light-driven carbon dioxide reduction to methane by nitrogenase in a photosynthetic bacterium. Proc Natl Acad Sci U S A, 2016. 113(36): p. 10163-7.
3. Brown, K.A., et al., Light-driven dinitrogen reduction catalyzed by a CdS:nitrogenase MoFe protein biohybrid. Science, 2016. 352(6284): p. 448-50.
4. Kuypers, M.M.M., H.K. Marchant, and B. Kartal, The microbial nitrogen-cycling network. Nat Rev Microbiol, 2018. 16(5): p. 263-276.
5. Wei, W., et al., A surface-display biohybrid approach to light-driven hydrogen production in air. Sci Adv, 2018. 4(2): p. eaap9253.
6. Wang, X., et al., Using synthetic biology to distinguish and overcome regulatory and functional barriers related to nitrogen fixation. PLoS One, 2013. 8(7): p. e68677.
7. Yang, J., et al., Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity. Proc Natl Acad Sci U S A, 2017. 114(12): p. E2460-E2465.
8. Yang, J., et al., Polyprotein strategy for stoichiometric assembly of nitrogen fixation components for synthetic biology. Proc Natl Acad Sci U S A, 2018. 115(36): p. E8509-E8517.
9. Yang, J.G., et al., Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 2014. 111(35): p. E3718-E3725.
10. Howard, J.B. and D.C. Rees, Structural basis of biological nitrogen fixation. Chemical Reviews, 1996. 96(7): p. 2965-2982.