Difference between revisions of "Part:BBa K1796007"

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	<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>	+
−	<h2>IGEM2018_Nanjing-China improve</h2>	+
−	<p>Based on the existing part, BBa_K1796007,  which is an essential component from <em>Paenibacillus sp.</em> WLY78&rsquo;s nitrogen  fixation gene cluster: <em>nif</em> Promoter,<em> nifB, nifH, nifD, nifK, nifE, nifN, nifX,  hesA, nifV,</em> We choose a new nitrogen fixation gene cluster from a more common strain <em>Paenibacillus polymyxa</em> CR1 and make some improvements, to comprise the nitrogen fixation system in our project. </p>	+
−	<p>Firstly, Because of existence of the  illegal <em>PstI</em> sites and <em>EcoRI</em> sites, the original gene sequence from <em>Paenibacillus polymyxa</em> 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 <em>nifB</em> to get them. The new part is RFC10 compatible which ensures a greater diversity when  designing synthetic biology projects.</p>	+
−	<p>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 N<sub>2</sub>(nitrogen) to NH<sub>3</sub>(ammonia). So certainly we need to test the nitrogen fixation’s heterologous expression level in <em>E.coli</em> to make sure the efficiency of photocatalytic nitrogen fixation.</p>	+
−	<p>[[File:T--Nanjing-China--011part-design.png|800px|thumb|center|Fig 1. Design of our project: Engineered E. coli cells with nitrogenase]] </p>	+
−	<p>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(<strong>Fig 2</strong>).</p>	+
−	[[File:T--Nanjing-China--11part.png|600px|thumb|center|Fig 2：Expression efficiency of Pnif]]	+
−	<p><font size="-1">Fig 2：Expression efficiency of Pnif</font></p></div>	+
−	<p>Comparison of the expression efficiency of  Pnif and T5 (IPTG Inducible) Promoter. <br />	+
−	T5 (IPTG Inducible) Promoter BBa_M50075; Pnif: nif promoter BBa_K1796001.</p>	+
−	<p>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 <strong>Fig3</strong>.</p>	+
−	[[File:T--Nanjing-China--QPCR1.jpg|500px|thumb|center]]<br />	+
−	[[File:T--Nanjing-China--QPCR2.jpg|500px|thumb|center|Fig 3. The qPCR results for components of nitrogen fixation system]]	+
−	<p align="left">From the results of qPCR we have known that not only the nitrogen gene cluster can  successfully heterologously expressed in the engineered <em>E. coli </em>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 <em>nif</em> 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.<br />	+
−	<a href="http://2018.igem.org/Team:Nanjing-China/Model">http://2018.igem.org/Team:Nanjing-China/Model</a> </p>	+
−	<p align="left">The improvements above have facilitate our team to accomplish our project and we sincerely wish it can help other use the gene cluster. </p>	+

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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

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.

 

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