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.
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
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 |