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.
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.
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.
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.
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（Fig 3).
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.
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.
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.
//chassis/prokaryote/ecoli
| proteins | |
| strain | Paenibacillus polymyxa CR1 |