Difference between revisions of "Part:BBa K1796007"

(→‎Parametre of Protein)
 
(10 intermediate revisions by 2 users not shown)
Line 24: Line 24:
 
<p> Theoretical pI: 6.64 </p>
 
<p> Theoretical pI: 6.64 </p>
 
<p> </p>
 
<p> </p>
−
<p> Amino acid composition:聽 </p>
+
<p> Amino acid composition: </p>
 
<p> </p>
 
<p> </p>
 
<p> Ala (A)  46      9.2% </p>
 
<p> Ala (A)  46      9.2% </p>
Line 98: Line 98:
 
<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>
−
<p> The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro). </p>
+
<p> The estimated half-life is: 30 hours (mammalian reticulocytes, in vitr
−
<p>                             >20 hours (yeast, in vivo). </p>
+
-->Nanjing-China2018
−
<p>                             >10 hours (Escherichia coli, in vivo). </p>
+
 
−
<p> </p>
+
<h2>IGEM2018_Nanjing-China  improve </h2>
−
<p> </p>
+
<p>The existing part, BBa_K1796007, is an essential component of the <em>Paenibacillus  sp.</em> WLY78&rsquo;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:</p>
−
<p> Instability index:The instability index (II) is computed to be 43.03 </p>
+
  <p>(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.</p>
−
<p> This classifies the protein as unstable. </p>
+
  <p>(2) The complete genome of <em>Paenibacillus polymyxa</em> CR1 has  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>
−
<p> </p>
+
<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>
−
<p> </p>
+
<p>&nbsp;</p>
−
<p> </p>
+
<p>[[File:T--Nanjing-China--1%2B2.jpg|800px|thumb|center|Figure 1a）Engineered E. coli cells with nitrogenase<br />
−
<p> Aliphatic index: 87.96 </p>
+
1b）Fluorescence intensity detemination]] </p>
−
<p> </p>
+
<p> [[File:T--Nanjing-China--qRT-PCR.jpg|600px|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>
−
<p> Grand average of hydropathicity (GRAVY): -0.253 </p>
+
<p align="left">Hopefully,  the aforementioned improvements and relevant testing results can facilitate the  utilization of the improved part for other iGEM team.   </p>
−
<p> Aliphatic index: 92.50 </p>
+
−
<p> </p>
+
−
<p> Grand average of hydropathicity (GRAVY): -0.161 </p>
+

Latest revision as of 11:37, 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 -->Nanjing-China2018

IGEM2018_Nanjing-China improve

<p>The existing part, BBa_K1796007, is an essential component of the Paenibacillus sp. WLY78’s nitrogen fixation gene (nif) cluster arranged in the order of nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, nifV. Instead of directly cloning WLY78 nifB using BBa_K1796007 as the template, a minimal nif cluster from Paenibacillus polymyxa CR1 that also contained nifB (nucleoid acid sequence similarity 96% as compared to WLY78 nifB) was chemically synthesized and incorporated into commercially available cloning vector pUC57. After that, CR1 nifB was obtained by PCR amplification using pUC57-nif as the template and subsequently introduced into pSB1C3 backbone through restriction enzyme digestion. Below, we discuss why we made such an improvement:

(1) Both nifB 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.

(2) The complete genome of Paenibacillus polymyxa CR1 has 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 nif cluster, nifB of a bacterium with clear genetic background, such as Paenibacillus polymyxa CR1, may be more valuable for researchers of relevant field.

In addition, to test whether the nifB could express in gram-negative E. coli JM109</a> as a part of the nif cluster, 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, Pnif 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, 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.

Hopefully, the aforementioned improvements and relevant testing results can facilitate the utilization of the improved part for other iGEM team.