Difference between revisions of "Part:BBa K2740012"

Revision as of 11:06, 13 October 2018

CR1 nifB

CR1 nifB encodes nitrogen fixation protein NifB that is essential for biosynthesis of the active-site nitrogenase cofactor. If the CR1 nifB was deleted, the nitrogen fixation would not happen.

Sequence and Features

Parameter of Protein

Number of amino acids: 499

Molecular weight: 54885.93

Theoretical pI: 7.24

Amino acid composition:
Ala (A)  45    9.0%
Arg (R)  35    7.0%
Asn (N)  18   3.6%
Asp (D)  23   4.6%
Cys (C)  16    3.2%
Gln (Q)  19    3.8%
Glu (E)  38    7.6%
Gly (G)  42    8.4%
His (H)  17    3.4%
Ile (I)    29   5.8%
Leu (L)  41    8.2%
Lys (K)  26    5.2%
Met (M)  12   2.4%
Phe (F)  13    2.6%
Pro (P)  25     5.0%
Ser (S)  28     5.6%
Thr (T)  16    3.2%
Trp (W)  2     0.4%
Tyr (Y)  13    2.6%
Val (V)  41    8.2%
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): 61
Total number of positively charged residues (Arg + Lys): 61

Atomic composition:

Carbon      C          2398
Hydrogen    H         3853
Nitrogen    N            703
Oxygen      O          716
Sulfur      S              28

Formula: C2398H3853N703O716S28
Total number of atoms: 7698

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 43.00
This classifies the protein as unstable.

Aliphatic index: 87.56

Grand average of hydropathicity (GRAVY): -0.254

Nitrogenase is a complex enzyme system consisting of nine protein components.Additionally, to maintain stoichiometry of these protein components is an essential requirement for nitrogenase biosynthesis and activity. However, there is only one copy of each structure gene present in the nif gene cluster. Therefore, cloning each of these nif genes and setting as independent part can facilitate the regulation of balancing expression ratios from the transcription and/or translation level(s) when they are heterogeneously expressed in non-diazotrophic hosts.We sent the sequences of the PCR template 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.

Molecular modeling of nifB

To learn more about the molecular structure of nitrogenase reductase NifB encoded by nifB, we use Swiss-Model to get the molecular model of the protein encoded by nifB, which  is essential for biosynthesis of the active-site nitrogenase cofactor.

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).

[[File:T--Nanjing-China--11part.png|400px|thumb|center|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.
http://2018.igem.org/Team:Nanjing-China/Model

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

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. And CR1 nifB is an essential component of nitrogen fixation system.