Difference between revisions of "Part:BBa K2740011"

Latest revision as of 10:39, 16 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

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

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

Figure 1a）Engineered E. coli cells with nitrogenase
1b）Fluorescence intensity detemination

Figure 2. Expression profiles of each structure gene in the nif cluster that overexpressed in engineered E.coli JM109 (EJNC). E.coli JM109 (EJ) severs as control and relative expression compared to the housekeeping gene 16S rRNA is shown. N.D. represent not ditected.

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.