Difference between revisions of "Part:BBa K1796007"

Revision as of 14:37, 12 October 2018

nifB from Paenibacillus sp. WLY78

essential for biosynthesis of the active-site nitrogenase cofactor and encodes a radical A-adenosylmethionine(SAM)-dependent enzyme that inserts the central carbon atom into the eight-Fe core of nifB cofactor(nifB-co).

Sequence and Features

Parameter of Protein

Number of amino acids: 499

Molecular weight: 54869.8

Theoretical pI: 6.64

Amino acid composition:

Ala (A) 46 9.2%

Arg (R) 35 7.0%

Asn (N) 17 3.4%

Asp (D) 23 4.6%

Cys (C) 16 3.2%

Gln (Q) 19 3.8%

Glu (E) 40 8.0%

Gly (G) 42 8.4%

His (H) 17 3.4%

Ile (I) 30 6.0%

Leu (L) 41 8.2%

Lys (K) 25 5.0%

Met (M) 11 2.2%

Phe (F) 13 2.6%

Pro (P) 25 5.0%

Ser (S) 27 5.4%

Thr (T) 17 3.4%

Trp (W) 2 0.4%

Tyr (Y) 13 2.6%

Val (V) 40 8.0%

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): 63

Total number of positively charged residues (Arg + Lys): 60

Atomic composition:Carbon C 2398

Hydrogen H 3849

Nitrogen N 701

Oxygen O 719

Sulfur S 27

Formula: C2398H3849N701O719S27Total number of atoms: 7694

Extinction coefficients:Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.

Ext. coefficient 31370

Abs 0.1% (=1 g/l) 0.572, assuming all pairs of Cys residues form cystines

Ext. coefficient 30370

Abs 0.1% (=1 g/l) 0.553, 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 43.03

This classifies the protein as unstable.

Aliphatic index: 87.96

Grand average of hydropathicity (GRAVY): -0.253

Aliphatic index: 92.50

Grand average of hydropathicity (GRAVY): -0.161

IGEM2018_Nanjing-China improve

Based on the existing part, BBa_K1796007, which is an essential component from Paenibacillus sp. WLY78’s nitrogen fixation gene cluster: nif Promoter, nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, nifV, We choose a new nitrogen fixation gene cluster from a more common strain Paenibacillus polymyxa CR1 and make some improvements, to comprise the nitrogen fixation system in our project.

Firstly, Because of existence of the illegal PstI sites and EcoRI sites, the original gene sequence from Paenibacillus polymyxa CR1 and the existing part, BBa_K1796007 is not RFC10 compatible, which is not convenient for us and other teams to use this part. So to make the part easier to operate, we make some synonymous mutations to reform the gene sequence and chemically synthesize the entire nitrogen fixation gene cluster, then we can PCR then isolated gene gene or basic part like nifB to get them. The new part is RFC10 compatible which ensures a greater diversity when designing synthetic biology projects.

Secondly, in our this year’s project, we intends to establish a sound and ideal whole-cell photocatalytic nitrogen fixation system. And we use the engineered E. coli cells to express nitrogenases(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). So certainly we need to test the nitrogen fixation’s heterologous expression level in E.coli to make sure the efficiency of photocatalytic nitrogen fixation.

Fig 1. Design of our project: Engineered E. coli cells with nitrogenase

In order to test the expression efficiency of the nif cluster,firstly we measured the transcriptional activity of nif promoter by combining it with the gene of fluorescent protein Dronpa,with T5 (IPTG Inducible) Promoter, BBa_M50075 as a positive control(Fig 2).

Fig 2：Expression efficiency of Pnif

Fig 2：Expression efficiency of Pnif

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

As demonstrated above, nif promoter is quite strong,however, how capable it is in our nitrogen fixation system remains an unclear question. So we also detected the expression level of the essential components in our system by conducting Real-time Quantitative PCR(QPCR),using 16S DNA as an internal reference.The results are shown in Fig3.

Fig 3. The qPCR results for components of nitrogen fixation system

From the results of qPCR we have known that not only the nitrogen gene cluster can successfully heterologously expressed in the engineered E. coli and but also the relative transcriptional level of each component of nitrogen gene cluster is different. Based on these analysis, our team created a mathematical model to optimize the arrangement of the nif gene cluster. This model helped we optimized our design and provided some new perspectives of our nitrogen-fixation system in transcriptional level. And you can see the detailed model by clicking the following link.
<a href="http://2018.igem.org/Team:Nanjing-China/Model">http://2018.igem.org/Team:Nanjing-China/Model</a>

The improvements above have facilitate our team to accomplish our project and we sincerely wish it can help other use the gene cluster.