Difference between revisions of "Part:BBa K2740011"
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   <h2>Confirmation of Expression of Nitrogen  Fixation Gene Cluster</h2>
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   <h2>Improve:Confirmation of Expression of Nitrogen  Fixation Gene Cluster</h2>
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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>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(Fig 1) 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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Revision as of 13:33, 7 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.

Nanjing_China 's Measurement: Expression efficiency of Pnif

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.

T--Nanjing-China--11part.png

Comparison of the expression efficiency of Pnif and T5 (IPTG Inducible) Promoter.
T5 (IPTG Inducible) Promoter BBa_M50075; Pnif: nif promoter BBa_K1796001.

 

Improve:Confirmation of Expression of Nitrogen Fixation Gene Cluster

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 Paenibacillus sp. WLY78. We choose a new nitrogen fixation gene cluster from more common strain Paenibacillus polymyxa CR1, to comprise the nitrogen fixation system in our project.

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(Fig 1) 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.