View source for Part:BBa K2740012


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<partinfo>BBa_K2740012 short</partinfo>

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.

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===Usage and Biology===
BBa K2740012

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<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K2740012 SequenceAndFeatures</partinfo>


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===Functional Parameters===
<partinfo>BBa_K2740012 parameters</partinfo>
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<h2><u>Parameter of Protein</u></h2>
<p align="left">&nbsp;</p>
<p align="left">Number  of amino acids: 499</p>
<p align="left">Molecular  weight: 54885.93</p>
<p align="left">Theoretical  pI: 7.24</p>
<p align="left">Amino  acid composition: <br />
  Ala  (A)  45    9.0%<br />
  Arg  (R)  35    7.0%<br />
  Asn  (N)  18   3.6%<br />
  Asp  (D)  23   4.6%<br />
  Cys  (C)  16    3.2%<br />
  Gln  (Q)  19    3.8%<br />
  Glu  (E)  38    7.6%<br />
  Gly  (G)  42    8.4%<br />
  His  (H)  17    3.4%<br />
  Ile  (I)    29   5.8%<br />
  Leu  (L)  41    8.2%<br />
  Lys  (K)  26    5.2%<br />
  Met  (M)  12   2.4%<br />
  Phe  (F)  13    2.6%<br />
  Pro  (P)  25     5.0%<br />
  Ser  (S)  28     5.6%<br />
  Thr  (T)  16    3.2%<br />
  Trp  (W)  2     0.4%<br />
  Tyr  (Y)  13    2.6%<br />
  Val  (V)  41    8.2%<br />
  Pyl  (O)   0     0.0%<br />
  Sec  (U)   0    0.0%</p>
<p align="left"> (B)   0          0.0%<br />
  (Z)   0   0.0%<br />
  (X)   0          0.0%</p>
<p align="left">&nbsp;</p>
<p align="left">Total  number of negatively charged residues (Asp + Glu): 61<br />
  Total  number of positively charged residues (Arg + Lys): 61</p>
<p align="left">Atomic  composition:</p>
<p align="left">Carbon      C          2398<br />
  Hydrogen    H         3853<br />
  Nitrogen    N            703<br />
  Oxygen      O          716<br />
  Sulfur      S              28</p>
<p align="left">Formula:  C2398H3853N703O716S28<br />
  Total  number of atoms: 7698</p>
<p align="left">Extinction  coefficients:</p>
<p align="left">Extinction  coefficients are in units of  M-1 cm-1,  at 280 nm measured in water.</p>
<p align="left">Ext.  coefficient    31370<br />
  Abs  0.1% (=1 g/l)   0.572, assuming all pairs  of Cys residues form cystines</p>
<p align="left">&nbsp;</p>
<p align="left">Ext.  coefficient    30370<br />
  Abs  0.1% (=1 g/l)   0.553, assuming all Cys  residues are reduced</p>
<p align="left">Estimated  half-life:</p>
<p align="left">The  N-terminal of the sequence considered is M (Met).</p>
<p align="left">The  estimated half-life is: 30 hours (mammalian reticulocytes, in vitro).<br />
  &gt;20 hours  (yeast, in vivo).<br />
  &gt;10 hours  (Escherichia coli, in vivo).</p>
<p align="left">&nbsp;</p>
<p align="left">Instability  index:</p>
<p align="left">The  instability index (II) is computed to be 43.00<br />
  This  classifies the protein as unstable.</p>
<p align="left">&nbsp;</p>
<p align="left">Aliphatic  index: 87.56</p>
<p align="left">Grand  average of hydropathicity (GRAVY): -0.254</p>
<div>
  <h2>Design Notes</h2>
</div>
<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.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.</p>
<h2>Molecular modeling of nifB</h2>
<p align="left">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.</p>
<p>[[File:T--Nanjing-China--nifB-structure.png|400px|thumb|center]]</p>
<h2>IGEM2018_Nanjing-China  improve </h2>
<p>The existing part, BBa_K1796007, is an essential component of the <em>Paenibacillus  sp.</em> WLY78&rsquo;s nitrogen fixation gene (<em>nif</em>)  cluster arranged in the order of <em>nif</em>B, <em>nif</em>H, <em>nif</em>D, <em>nif</em>K, <em>nif</em>E, <em>nif</em>N, <em>nif</em>X, <em>hes</em>A, <em>nif</em>V. Instead of directly cloning WLY78 <em>nif</em>B using BBa_K1796007 as the template, a minimal <em>nif</em> cluster from <em>Paenibacillus  polymyxa</em> CR1 that also contained <em>nif</em>B  (nucleoid acid sequence similarity 96% as compared to WLY78 <em>nif</em>B) was chemically synthesized and  incorporated into commercially available cloning vector pUC57. After that, CR1 <em>nif</em>B was obtained by PCR amplification  using pUC57-<em>nif</em> as the template and  subsequently introduced into pSB1C3 backbone through restriction enzyme  digestion. Below, we discuss why we made such an improvement:<br />
  (1) Both <em>nif</em>B 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.<br />
  (2) The complete genome of <em>Paenibacillus polymyxa</em> 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 <em>nif</em> cluster, <em>nif</em>B of a bacterium with clear genetic  background, such as<em> Paenibacillus polymyxa</em> CR1, may be more valuable for  researchers of relevant field. </p>
<p>In addition, to test whether the <em>nif</em>B  could express in gram-negative <em>E. coli</em> JM109 as a part of the <em>nif</em> cluster,  pUC57-<em>nif </em>was inreoduced into JM109  via electroporation (Figure 1a). But before qRT-PCR determination, the function  and strength of the native promoter in <em>nif</em> cluster (P<em>nif</em>) 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, P<em>nif </em>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, P<em>nif</em> was strong enough to drive the expression of each structure  gene in the <em>nif</em> cluster including <em>nif</em>B though with different relative  expression level.</p>
<p>[[File:T--Nanjing-China--1%2B2.jpg|800px|thumb|center|Figure 1a）Engineered E. coli cells with nitrogenase<br />
1b）Fluorescence intensity detemination]] </p>
 <p> [[File:T--Nanjing-China--qRT-PCR.jpg|800px|thumb|center|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.]]</p>

<h2>Usage</h2>
  <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. And CR1 nifB is an essential component of nitrogen fixation system.</p>
<h2>Reference</h2>
  <p>1.             Wang, L., et al., <em>A minimal nitrogen fixation gene cluster  from Paenibacillus sp. WLY78 enables expression of active nitrogenase in  Escherichia coli.</em> PLoS Genet, 2013. <strong>9</strong>(10):  p. e1003865.<br />
    2.             Fixen, K.R., et  al., <em>Light-driven carbon dioxide  reduction to methane by nitrogenase in a photosynthetic bacterium.</em> Proc  Natl Acad Sci U S A, 2016. <strong>113</strong>(36):  p. 10163-7.<br />
    3.             Brown, K.A., et  al., <em>Light-driven dinitrogen reduction  catalyzed by a CdS:nitrogenase MoFe protein biohybrid.</em> Science, 2016. <strong>352</strong>(6284): p. 448-50.<br />
    4.             Kuypers, M.M.M.,  H.K. Marchant, and B. Kartal, <em>The  microbial nitrogen-cycling network.</em> Nat Rev Microbiol, 2018. <strong>16</strong>(5): p. 263-276.<br />
    5.             Wei, W., et al., <em>A surface-display biohybrid approach to  light-driven hydrogen production in air.</em> Sci Adv, 2018. <strong>4</strong>(2): p. eaap9253.<br />
    6.             Wang, X., et  al., <em>Using synthetic biology to  distinguish and overcome regulatory and functional barriers related to nitrogen  fixation.</em> PLoS One, 2013. <strong>8</strong>(7):  p. e68677.<br />
    7.             Yang, J., et  al., <em>Modular electron-transport chains  from eukaryotic organelles function to support nitrogenase activity.</em> Proc  Natl Acad Sci U S A, 2017. <strong>114</strong>(12):  p. E2460-E2465.<br />
    8.             Yang, J., et  al., <em>Polyprotein strategy for  stoichiometric assembly of nitrogen fixation components for synthetic biology.</em> Proc Natl Acad Sci U S A, 2018. <strong>115</strong>(36):  p. E8509-E8517.<br />
    9.             Yang, J.G., et  al., <em>Reconstruction and minimal gene  requirements for the alternative iron-only nitrogenase in Escherichia coli.</em> Proceedings of the National Academy of Sciences of the United States of  America, 2014. <strong>111</strong>(35): p.  E3718-E3725.<br />
    10.          Howard, J.B. and  D.C. Rees, <em>Structural basis of biological  nitrogen fixation.</em> Chemical Reviews, 1996. <strong>96</strong>(7): p. 2965-2982.</p>