Difference between revisions of "Part:BBa K2740011"

Revision as of 05:49, 4 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

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

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.

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.

alt text

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