Part:BBa_K2740011
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
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE 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 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE 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.
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.
//chassis/prokaryote/ecoli
| proteins | |
| strain | Paenibacillus polymyxa CR1 |