Difference between revisions of "Part:BBa K2740018"

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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>
 
<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>
 +
<h2>Molecular modeling of nifX</h2>
 +
<p align="left">To  learn more about the molecular structure of nitrogen fixation protein NifX that  favors the insertion of molybdenum-iron protein cofactors into nitrogenase  encoded by nifX, we use Swiss-Model to get the molecular model.</p>
 +
[[File:T--Nanjing-China--nifX-structure.png|800px|thumb|center]]
 +
<h2>Confirmation of Expression of nifX</h2>
 +
<p align="left">To  verify the expression of nitrogenase gene, we conducted Real-time Quantitative  PCR(QPCR) to detect the transcription level of <em>nif</em> gene cluster in engineered <em>E.  coli</em>, using 16S DNA as an internal reference. The result provided the  relative expression level of each <em>nif</em>X  in our constructed <em>E. coli </em>strain. </p>
 +
[[File:T--Nanjing-China--nifX-1.png|800px|thumb|center]]<br/>
 +
[[File:T--Nanjing-China--nifX-2.png|800px|thumb|center]]
 +
<p>From the results of qPCR, we know the nifX  gene in engineered <em>E. coli relatively  fractionally expressed.</em></p>
 +
<div>
 +
  <h2>Usage</h2>
 +
</div>
 +
<p>In our this year&rsquo;s project, we intends to  establish a sound and ideal whole-cell photocatalytic nitrogen fixation system.  We use the engineered <em>E. coli</em> 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 <em>Paenibacillus polymyxa</em> CR1 to test its expression  level.</p>
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<p>&nbsp;</p>
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<p align="left">&nbsp;</p>

Revision as of 03:11, 8 October 2018


CR1 nifX

CR1 nifX encodes nitrogen fixation protein NifX that favors the insertion of molybdenum-iron protein cofactors into nitrogenase.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Parameter of Protein

Number of amino acids: 129

Molecular weight: 14126.33

Theoretical pI: 6.51

Amino acid composition:
Ala (A)  16  12.4%
Arg (R)   7    5.4%
Asn (N)   4   3.1%
Asp (D)   3   2.3%
Cys (C)   1    0.8%
Gln (Q)   7    5.4%
Glu (E)  11    8.5%
Gly (G)  10    7.8%
His (H)   5    3.9%
Ile (I)    10   7.8%
Leu (L)  10    7.8%
Lys (K)   6    4.7%
Met (M)  5     3.9%
Phe (F)   7     5.4%
Pro (P)   4     3.1%
Ser (S)   7     5.4%
Thr (T)   5     3.9%
Trp (W)  1     0.8%
Tyr (Y)   0    0.0%
Val (V)  10    7.8%
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): 14
Total number of positively charged residues (Arg + Lys): 13

Atomic composition:

Carbon      C           627
Hydrogen    H         1006
Nitrogen    N            178
Oxygen      O          181
Sulfur      S               6

Formula: C627H1006N178O181S6
Total number of atoms: 1998

Extinction coefficients:

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

Ext. coefficient     5500
Abs 0.1% (=1 g/l)   0.389, assuming all pairs of Cys residues form cystines

 

Ext. coefficient     5500
Abs 0.1% (=1 g/l)   0.389, 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 47.67
This classifies the protein as unstable.

 

Aliphatic index: 95.35

Grand average of hydropathicity (GRAVY): 0.051

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 nifX

To learn more about the molecular structure of nitrogen fixation protein NifX that favors the insertion of molybdenum-iron protein cofactors into nitrogenase encoded by nifX, we use Swiss-Model to get the molecular model.

T--Nanjing-China--nifX-structure.png

Confirmation of Expression of nifX

To verify the expression of nitrogenase gene, we conducted Real-time Quantitative PCR(QPCR) to detect the transcription level of nif gene cluster in engineered E. coli, using 16S DNA as an internal reference. The result provided the relative expression level of each nifX in our constructed E. coli strain.

T--Nanjing-China--nifX-1.png

T--Nanjing-China--nifX-2.png

From the results of qPCR, we know the nifX gene in engineered E. coli relatively fractionally expressed.

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.