Difference between revisions of "Part:BBa K2740011"
 
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<partinfo>BBa_K2740011 parameters</partinfo>
 
<partinfo>BBa_K2740011 parameters</partinfo>
 
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   <h2>Parameter of Protein </h2>
 
   <h2>Parameter of Protein </h2>
 
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<p>It is a minimal gene cluster that can achieve nitrogen fixation when it is heterogeneously expressed. Therefore,  it can serve as a &ldquo; nitrogen fixation module&rdquo; and be introduced into non-diazotrophs to confer them with nitrogen  fixation capacity.Benefit from its compact structure (nine  genes) and relatively small size (10.5 kb), it can facilitate relevant genetic  manipulation. We sent the sequences 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>
 
<p>It is a minimal gene cluster that can achieve nitrogen fixation when it is heterogeneously expressed. Therefore,  it can serve as a &ldquo; nitrogen fixation module&rdquo; and be introduced into non-diazotrophs to confer them with nitrogen  fixation capacity.Benefit from its compact structure (nine  genes) and relatively small size (10.5 kb), it can facilitate relevant genetic  manipulation. We sent the sequences 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>
 
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   <h2>Nanjing_China 's Measurement: Expression  efficiency of Pnif</h2>
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   <h2>Confirmation of Expression  of&nbsp;Nitrogen Fixation Gene Cluster </h2>
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To test whether the nitrogen fixation gene cluster  could express in gram-negative <em>E. coli</em>JM109 , 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
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<p>[[File:T--Nanjing-China--1%2B2.jpg|800px|thumb|center|Figure 1a）Engineered E. coli cells with nitrogenase<br />
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  <h2>Improve: Confirmation of Expression of  nitrogen fixation gene cluster</h2>
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1b）Fluorescence intensity detemination]] </p>
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  <p>Based on the existing part complete line of nif cluster, BBa_K1796015, which contains essential components for nitrogen fixation: nif  Promoter, nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, nifV from the <em>Paenibacillus  sp.</em> WLY78. We choose a new nitrogen fixation gene cluster from more common  strain <em>Paenibacillus polymyxa</em> CR1, to comprise the nitrogen fixation system in our project.</p>
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  <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>
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  <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(<strong>Fig 1</strong>) 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.</p>
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  <p>[[File:T--Nanjing-China--011part-design.png|800px|thumb|center|Fig 1. Design of our project: Engineered E. coli cells with nitrogenase]] </p>
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  <p>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 compared with the BBa_K1796015 from Paenibacillus sp. WLY78.</p>
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  <p align="left">&nbsp;</p>
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<p>To make sure the expression efficiency of the nif cluster, at first we want to measure the feature the nif promoter. So we recombine the  Pnif(nif promoter) with the gene of fluorescent protein Dronpa with Pnif to  investigate the activity of Pnif tanscription activity. And we choose the T5  (IPTG Inducible) Promoter BBa_M50075 as a positive control.</p>
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[[File:T--Nanjing-China--11part.png|800px|thumb|center|Fig 2：Expression efficiency of Pnif]]
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<p>Comparison of the expression efficiency of Pnif and T5 (IPTG  Inducible) Promoter. <br />
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T5 (IPTG Inducible) Promoter BBa_M50075; Pnif: nif promoter  BBa_K1796001.</p>
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<p align="left">&nbsp;</p>
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<p>Although the nif promoter has been tested  as a quite strong promoter above, also we need to detect the expression level  of the essential components in our nitrogen fixation system. 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 nif gene in our constructed E. coli strain（<strong>Fig 3</strong>). <br />
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  After we compare the result with the ideal  expression ratio in Paenibacillus CR1 and model the transcription, we plan to optimize the nif gene cluster by adding promoters or altering the position of  genes.</p>
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  [[File:T--Nanjing-China--QPCR1.jpg|800px|thumb|center]]<br />
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  [[File:T--Nanjing-China--QPCR2.jpg|800px|thumb|center|Fig 3. The qPCR results for components of nitrogen fixation system]]
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  <p>Nitrogenase can not only reduce dinitrogen  to ammonia but also reduce ethylene to acetylene. Therefore, we use gas  chromatography to detect the amount of acetylene reduced, and indirectly detect  its nitrogen fixation activity. </p>
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   <h2>Usage</h2>
 
   <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.</p>
 
   <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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Latest revision as of 10:39, 16 October 2018


Nitrogen fixation (nif) gene cluster of Paenibacillus polymyxa CR1

A gene cluster enables synthesis of catalytically active nitrogenase in wild type P. polymyxa CR1 and the accordingly genetically engineered E. coli. This cluster is organized as an operon comprising nine structure genes nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV. Besides, it contains the native promoter that located upstream of nifB and the native terminator that located downstream nifV respectively.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 875
    Illegal PstI site found at 2887
    Illegal PstI site found at 6767
    Illegal PstI site found at 6925
    Illegal PstI site found at 7684
    Illegal PstI site found at 7878
    Illegal PstI site found at 10071
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 875
    Illegal PstI site found at 2887
    Illegal PstI site found at 6767
    Illegal PstI site found at 6925
    Illegal PstI site found at 7684
    Illegal PstI site found at 7878
    Illegal PstI site found at 10071
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 875
    Illegal PstI site found at 2887
    Illegal PstI site found at 6767
    Illegal PstI site found at 6925
    Illegal PstI site found at 7684
    Illegal PstI site found at 7878
    Illegal PstI site found at 10071
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 875
    Illegal PstI site found at 2887
    Illegal PstI site found at 6767
    Illegal PstI site found at 6925
    Illegal PstI site found at 7684
    Illegal PstI site found at 7878
    Illegal PstI site found at 10071
    Illegal NgoMIV site found at 4796
    Illegal NgoMIV site found at 5482
    Illegal NgoMIV site found at 7371
    Illegal AgeI site found at 5855
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 5046
    Illegal BsaI.rc site found at 8842
    Illegal SapI.rc site found at 2330
    Illegal SapI.rc site found at 5429


Parameter of Protein

 

Number of amino acids: 3384

Molecular weight: 379594.64

Theoretical pI: 9.15

Amino acid composition:
Ala (A)  274         8.1%
Arg (R)  286         8.5%
Asn (N)  97   2.9%
Asp (D)  131  3.9%
Cys (C)  114         3.4%
Gln (Q)  114         3.4%
Glu (E)  206         6.1%
Gly (G)  256         7.6%
His (H)  77    2.3%
Ile (I)   210   6.2%
Leu (L)  290         8.6%
Lys (K)  150         4.4%
Met (M)  110  3.3%
Phe (F)  100   3.0%
Pro (P)  197   5.8%
Ser (S)  247   7.3%
Thr (T)  152   4.5%
Trp (W)  79   2.3%
Tyr (Y)  99    2.9%
Val (V)  195   5.8%
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): 337
Total number of positively charged residues (Arg + Lys): 436

Atomic composition:

Carbon      C         16785
Hydrogen    H        26576
Nitrogen    N           4836
Oxygen      O         4768
Sulfur      S             224

Formula: C16785H26576N4836O4768S224
Total number of atoms: 53189

Extinction coefficients:

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

Ext. coefficient   589135
Abs 0.1% (=1 g/l)   1.552, assuming all pairs of Cys residues form cystines

 

Ext. coefficient   582010
Abs 0.1% (=1 g/l)   1.533, 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 54.80
This classifies the protein as unstable.

 

Aliphatic index: 82.43

Grand average of hydropathicity (GRAVY): -0.244

Design Notes

It is a minimal gene cluster that can achieve nitrogen fixation when it is heterogeneously expressed. Therefore, it can serve as a “ nitrogen fixation module” and be introduced into non-diazotrophs to confer them with nitrogen fixation capacity.Benefit from its compact structure (nine genes) and relatively small size (10.5 kb), it can facilitate relevant genetic manipulation. We sent the sequences 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.

Confirmation of Expression of Nitrogen Fixation Gene Cluster

To test whether the nitrogen fixation gene cluster could express in gram-negative E. coliJM109 , 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, Pnifwas 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.