Difference between revisions of "Part:BBa K2740019"

Latest revision as of 10:39, 8 October 2018

CR1 hesA

CR1 hesA encodes a NAD/FAD-binding protein, HesA, which is involved in molybdopterin and thiamine biosynthesis. It plays an important role in nitrogen fixation and is responsive to the availability of molybdenum.

Sequence and Features

Parameter of Protein

Number of amino acids: 254

Molecular weight: 27871.11

Theoretical pI: 5.42

Amino acid composition:
Ala (A)  25    9.8%
Arg (R)  19    7.5%
Asn (N)   8   3.1%
Asp (D)  12   4.7%
Cys (C)   8    3.1%
Gln (Q)   8    3.1%
Glu (E)  19    7.5%
Gly (G)  26  10.2%
His (H)   6    2.4%
Ile (I)   14     5.5%
Leu (L)  27  10.6%
Lys (K)   5    2.0%
Met (M)  13   5.1%
Phe (F)   3     1.2%
Pro (P)  12     4.7%
Ser (S)   9     3.5%
Thr (T)  13    5.1%
Trp (W)  3     1.2%
Tyr (Y)   9    3.5%
Val (V)  15    5.9%
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): 31
Total number of positively charged residues (Arg + Lys): 24

Atomic composition:

Carbon      C          1212
Hydrogen    H         1941
Nitrogen    N            347
Oxygen      O          364
Sulfur      S              21

Formula: C1212H1941N347O364S21
Total number of atoms: 3885

Extinction coefficients:

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

Ext. coefficient    30410
Abs 0.1% (=1 g/l)   1.091, assuming all pairs of Cys residues form cystines

Ext. coefficient    29910
Abs 0.1% (=1 g/l)   1.073, 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 44.28
This classifies the protein as unstable.

Aliphatic index: 89.92

Grand average of hydropathicity (GRAVY): -0.088

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.

Improve: Confirmation of Expression of nitrogen fixation gene cluster

Based on the existing part complete line of nif cluster, BBa_K1796015, which contains essential components for nitrogen fixation: nif Promoter, nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, nifV from the Paenibacillus sp. WLY78. We choose a new nitrogen fixation gene cluster from more common strain Paenibacillus polymyxa CR1, to comprise the nitrogen fixation system in our project.

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(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). 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.

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

<img width="38" height="16" src="19_clip_image001.gif" />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 compared with the BBa_K1796015 from Paenibacillus sp. WLY78.

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(Fig 2).

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.

Although the nif promoter has been tested as a quite strong promoter above, also we need to detect the expression level of the essential components in our nitrogen fixation system. To verify the expression of nitrogenase gene, we conducted Real-time Quantitative PCR(QPCR) to detect the transcription level of nif gene cluster in engineered E. coli, using 16S DNA as an internal reference. The result provided the relative expression level of each nif gene in our constructed E. coli strain（Fig 3).
After we compare the result with the ideal expression ratio in Paenibacillus CR1 and model the transcription, we plan to optimize the nif gene cluster by adding promoters or altering the position of genes.

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

Nitrogenase can not only reduce dinitrogen to ammonia but also reduce ethylene to acetylene. Therefore, we use gas chromatography to detect the amount of acetylene reduced, and indirectly detect its nitrogen fixation activity.

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.