Difference between revisions of "Part:BBa K2740012"

Latest revision as of 11:39, 16 October 2018

CR1 nifB

CR1 nifB encodes nitrogen fixation protein NifB that is essential for biosynthesis of the active-site nitrogenase cofactor. If the CR1 nifB was deleted, the nitrogen fixation would not happen.

Sequence and Features

Parameter of Protein

Number of amino acids: 499

Molecular weight: 54885.93

Theoretical pI: 7.24

Amino acid composition:
Ala (A)  45    9.0%
Arg (R)  35    7.0%
Asn (N)  18   3.6%
Asp (D)  23   4.6%
Cys (C)  16    3.2%
Gln (Q)  19    3.8%
Glu (E)  38    7.6%
Gly (G)  42    8.4%
His (H)  17    3.4%
Ile (I)    29   5.8%
Leu (L)  41    8.2%
Lys (K)  26    5.2%
Met (M)  12   2.4%
Phe (F)  13    2.6%
Pro (P)  25     5.0%
Ser (S)  28     5.6%
Thr (T)  16    3.2%
Trp (W)  2     0.4%
Tyr (Y)  13    2.6%
Val (V)  41    8.2%
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): 61
Total number of positively charged residues (Arg + Lys): 61

Atomic composition:

Carbon      C          2398
Hydrogen    H         3853
Nitrogen    N            703
Oxygen      O          716
Sulfur      S              28

Formula: C2398H3853N703O716S28
Total number of atoms: 7698

Extinction coefficients:

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

Ext. coefficient    31370
Abs 0.1% (=1 g/l)   0.572, assuming all pairs of Cys residues form cystines

Ext. coefficient    30370
Abs 0.1% (=1 g/l)   0.553, 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 43.00
This classifies the protein as unstable.

Aliphatic index: 87.56

Grand average of hydropathicity (GRAVY): -0.254

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.

Molecular modeling of nifB

To learn more about the molecular structure of nitrogenase reductase NifB encoded by nifB, we use Swiss-Model to get the molecular model of the protein encoded by nifB, which  is essential for biosynthesis of the active-site nitrogenase cofactor.

IGEM2018_Nanjing-China improve

The existing part, BBa_K1796007, is an essential component of the Paenibacillus sp. WLY78’s nitrogen fixation gene (nif) cluster arranged in the order of nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, nifV. Instead of directly cloning WLY78 nifB using BBa_K1796007 as the template, a minimal nif cluster from Paenibacillus polymyxa CR1 that also contained nifB (nucleoid acid sequence similarity 96% as compared to WLY78 nifB) was chemically synthesized and incorporated into commercially available cloning vector pUC57. After that, CR1 nifB was obtained by PCR amplification using pUC57-nif as the template and subsequently introduced into pSB1C3 backbone through restriction enzyme digestion. Below, we discuss why we made such an improvement:
(1) Both nifB genes from WLY78 and CR1 contain unwanted restriction sites that can not meet the compatibility requirements of the iGEM Parts Guidelines. Therefore, elimination of these site through chemical synthesis is necessary.
(2) The complete genome of Paenibacillus polymyxa CR1 has been thoroughly sequenced and deposited in NCBI with the accession number CP006941.2. Considering that there exist some other genes possessing regulatory function for the nif cluster, nifB of a bacterium with clear genetic background, such as Paenibacillus polymyxa CR1, may be more valuable for researchers of relevant field.

In addition, to test whether the nifB could express in gram-negative E. coli JM109 as a part of the nif cluster, pUC57-nif was inreoduced into JM109 via electroporation (Figure 1a). But before qRT-PCR determination, the function and strength of the native promoter in nif cluster (Pnif) were firstly tested in JM109 by fusing Dronpa as the reporter. T5 promoter (BBa_M50075) severed as control. As shown in Figure 1b, compared with T5 promoter, Pnif was much stronger in driving the expression of RFP and its expression pattern was constitutive. Transcriptional analysis was carried out afterward. As shown in Figure 2, Pnif was strong enough to drive the expression of each structure gene in the nif cluster including nifB though with different relative expression level.

Figure 1a）Engineered E. coli cells with nitrogenase
1b）Fluorescence intensity detemination

Figure 2. 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. And CR1 nifB is an essential component of nitrogen fixation system.

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.