<p> Estimated half-life:The N-terminal of the sequence considered is M (Met). </p>
<p> Estimated half-life:The N-terminal of the sequence considered is M (Met). </p>
<p> </p>
<p> </p>
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<p> The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro). </p>
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<p> The estimated half-life is: 30 hours (mammalian reticulocytes, in vitr
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<p> >20 hours (yeast, in vivo). </p>
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<p> >10 hours (Escherichia coli, in vivo). </p>
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<p> </p>
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<p> </p>
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<p> Instability index:The instability index (II) is computed to be 43.03 </p>
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<p> This classifies the protein as unstable. </p>
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<p> </p>
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<p> </p>
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<p> </p>
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<p> Aliphatic index: 87.96 </p>
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<p> </p>
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<p> Grand average of hydropathicity (GRAVY): -0.253 </p>
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<p> Aliphatic index: 92.50 </p>
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<p> </p>
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<p> Grand average of hydropathicity (GRAVY): -0.161 </p>
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<h2>IGEM2018_Nanjing-China improve </h2>
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<p>The existing part, BBa_K1796007, is an essential component of the <em>Paenibacillus sp.</em> WLY78’s nitrogen fixation gene (<em>nif</em>) cluster arranged in the order of <em>nif</em>B, <em>nif</em>H, <em>nif</em>D, <em>nif</em>K, <em>nif</em>E, <em>nif</em>N, <em>nif</em>X, <em>hes</em>A, <em>nif</em>V. Instead of directly cloning WLY78 <em>nif</em>B using BBa_K1796007 as the template, a minimal <em>nif</em> cluster from <em>Paenibacillus polymyxa</em>CR1 that also contained <em>nif</em>B (nucleoid acid sequence similarity 96% as compared to WLY78 <em>nif</em>B) was chemically synthesized and incorporated into commercially available cloning vector pUC57. After that, CR1 <em>nif</em>B was obtained by PCR amplification using pUC57-<em>nif</em> as the template and subsequently introduced into pSB1C3 backbone through restriction enzyme digestion. Below, we discuss why we made such an improvement:<br />
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(1)Both <em>nif</em>B genes from WLY78 and CR1 contain unwanted restriction sites that can not meet the compatibility requirements of the iGEM Parts Guidelines. Therefore, elimination of these site through chemical synthesis is necessary.<br />
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(2) The complete genome of <em>Paenibacillus polymyxa</em> CR1has been thoroughly sequenced and deposited in NCBI with the accession number CP006941.2. Considering that there exist some other genes possessing regulatory function for the <em>nif</em> cluster, <em>nif</em>B of a bacterium with clear genetic background, such as<em> Paenibacillus polymyxa</em> CR1, may be more valuable for researchers of relevant field. </p>
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<p>In addition, to test whether the <em>nif</em>B could express in gram-negative <em>E. coli</em> JM109</a> as a part of the <em>nif</em> cluster, pUC57-<em>nif </em>was inreoduced into JM109 via electroporation (Figure 1a). But before qRT-PCR determination, the function and strength of the native promoter in <em>nif</em> cluster (P<em>nif</em>) 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, P<em>nif </em>was 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, P<em>nif</em> was strong enough to drive the expression of each structure gene in the <em>nif</em> cluster including <em>nif</em>B though with different relative expression level.</p>
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<p> </p>
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<p>[[File:T--Nanjing-China--1%2B2.jpg|800px|thumb|center|Figure 1a）Engineered E. coli cells with nitrogenase<br />
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1b）Fluorescence intensity detemination]] </p>
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<p> [[File:T--Nanjing-China--qRT-PCR.jpg|800px|thumb|center|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.]]</p>
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<p align="left">Hopefully, the aforementioned improvements and relevant testing results can facilitate the utilization of the improved part for other iGEM team. </p>
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Revision as of 11:28, 16 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
Assembly Compatibility:
10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 1156
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 1156
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 1156
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 1156
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 1156 Illegal AgeI site found at 129 Illegal AgeI site found at 1089 Illegal AgeI site found at 1444
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1261
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 vitr
