whole line of nif cluster,contain Pnif,nifBHDKENXV,hesA
NifH(BBa_K1796006) is taken from genomic sequence of Paenibacillus sp. WLY78, we apply promotions on the sequences and synthesised it.
Dinitrogenase reductase, important in nitrogen fixation.
We put it in a pET-28b plasmid to express it.
promoter of Paenibacillus sp. WLY78 nif gene cluster
Pnif is from Paenibacillus sp. WLY78 genome,located upstream of nifB,the first gene of nitrogen fixation cluster.
The promoter of Paenibacillus sp. WLY78 nif gene cluster, We named it Pnif. Pnif is a σ70-dependent promoter located upstream of nifB. Pnif is in control of all the gene clusters of nitrogen fixation genome. Confirmation of nif promoter We standardized the nif promoter of Paenibacillus sp. WLY78, and linked it to the upstream of 3 reporter genes (RFP reporter: BBa_K1357010, YFP reporter: BBa_E0430;amilCP reporter: BBa_K1357009) to confirm the reliability of this promoter. The results are shown in Figure 2. A B C http://2015.igem.org/wiki/images/b/b2/Confirmation_of_Pnif_AB.png http://2015.igem.org/wiki/images/thumb/3/34/C-confirmation_of_Pnif.jpeg/622px-C-confirmation_of_Pnif.jpeg Fig.4. Confirmation of nif promoter of Paenibacillus sp. WLY78. (A) Pnif+RFP reporter; (B) Pnif+YFP reporter;(C)Pnif+amilCP reporter. The results confirmed the reliability of Pnif. The initiation of Pnif does not require inducement. Thus it is a effective constitutive promoter.
effeciency of the promoter in E.coli for ordinary parts
NifB(BBa_K1796007) is taken from genomic sequence of Paenibacillus sp.WLY78, we apply promotions on the sequences and synthesised it.
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).
The part contains a sequence of 21bp possible RBS region
nifD promoted from Paenibacillus sp. WLY78
NifD(BBa_K1796008) is taken from genomic sequence of Paenibacillus sp.WLY78, we apply promotions on the sequences and synthesised it.
Function: αsubunits of dinitrogease which also called FeMo protein. NifD encode a metallocluster: FeMo-co, a [Mo-7Fe-9S-C-homocitrate] cluster which serves as the active site of substrate binding and reduction. We abtained the sequences from genomes of Paenibacillus sp. WLY78. First, we promoted it ourselves（done by Nan Wang and Nannan Xie).Then we sent the sequences to synthesis, but unfortunately, striction enzyme cut site was involved after they promoted it again. But we were not informed of the error in promotion.After several times failed expriments, we found the problem.We tackled them over the matter, mistakes were corrected, but the correct genes can't arrive on time,it can only arrive after the deadline. Sequence submit is promoted by the synthesis company, containing a PstI in the gene. The sequence we promoted: CAGGAGGGAGGAATAGGCCAAATGTCTTCTATCGTTGACAAAGGTAAACAGATCGTTGAAGAAATCCTGGAAGTTTACCCGAAAAAAGCTAAAAAAGACCGTACCAAACACTTCGAAATCGCTGACGAAGAACTGGTTAACTGCGGTACCTGCTCTATCAAATCTAACATGAAATCTCGTCCGGGTGTTATGACCGCTCGTGGTTGCGCTTACGCTGGTTCTAAAGGTGTTGTTTGGGGTCCGATCAAAGACATGGTTCACATCTCTCACGGTCCGATCGGTTGCGGTCAGTACTCTTGGGGTACCCGTCGTAACTACGCTAACGGTATCCTGGGTATCGACAACTTCACCGCTATGCAGATCACCTCTAACTTCCAGGAAAAAGACATCGTTTTCGGTGGTGACAAAAAACTGGAAGTTATCTGCCGTGAAATCAAAGAAATGTTCCCGCTGGCTAAAGGTATCTCTGTTCAGTCTGAATGCCCGGTTGGTCTGATCGGTGACGACATCGGTGCTGTTGCTAAAAAAATGACCGAAGAACTGGGTATCCCGGTTATCCCGGTTCGTTGCGAAGGTTTCCGTGGTGTTTCTCAGTCTCTGGGTCACCACATCGCTAACGACGCTATCCGTGACTTCCTGATGGGTCGTCGTGAACTGAAAGAATGCGGTCCGTACGACGTTTCTATCATCGGTGACTACAACATCGGTGGTGACGCTTGGGCTTCTCGTATCCTGCTGGAAGAAATGGGTCTGCGTGTTATCGCTCAGTGGTCTGGTGACGGTACCATCAACGAACTGGGTATCGCTCACAAATCTAAACTGAACCTGATCCACTGCCACCGTTCTATGAACTACATGTGCACCACCATGGAACAGGAATACGGTATCCCGTGGATGGAATACAACTTCTTCGGTCCGACCAAAACCATGGAATCTCTGCGTGCTATCGCTGCTCGTTTCGACGAAACCATCCAGGAAAAATGCGAACAGGTTATCGCTCAGTACATGCCGCAGATGGAAGCTGTTATCCGTAAATACCGTCCGCGTCTGGAAGGTAAAAAAGTTATGCTGCTGATCGGTGGTCTGCGTGCTCGTCACACCATCGGTGCTTACGAAGACCTGGGTATGGAAATCGTTGCTACCGGTTACGAATTTGCTCACAAAGACGACTACGAAAAAACCTTCCCGGACGTTAAAGAAGGTACCATCCTGTACGACGACCCGACCGCTTACGAACTGGAAGAACTGGCTCAGCGTCTGAACATCGACCTGATGGGTGCTGGTGTTAAAGAAAAATACGTTTACCACAAAATGGGTATCCCGTTCCGTCAGATGCACTCTTGGGACTACTCTGGTCCATACCACGGGTTTGACGGCTTCAAGATCTTCGCGCGTGACATGGACATGACCATCAACTCTCCGGTTTGGTCTCTGCTGCCGTCTCGTCAGACCGCTGAAGTTCCGGTTTAATAA
We abtained the sequences from genomes of Paenibacillus sp. WLY78. First, we promoted it ourselves（done by Nan Wang and Nannan Xie).Then we sent the sequences to synthesis, but unfortunately, striction enzyme cut site was involved after they promoted it again. But the part can be assembled by gibson assembly,that is what we did.
nifK promoted from Paenibacillus sp. WLY78
NifK(BBa_K1796009) is taken from genomic sequence of Paenibacillus sp.WLY78, we apply promotions on the sequences and synthesised it.
Function: β subunits of dinitrogease which also called FeMo protein.NifK encode a metallocluster: the P-cluster, a [8Fe-7S] cluster which shuttles electrons to FeMo-co. We abtained the sequences from genomes of Paenibacillus sp. WLY78. First, we promoted it ourselves（done by Nan Wang and Nannan Xie).Then we sent the sequences to synthesis.
We abtained the sequences from genomes of Paenibacillus sp. WLY78. First, we promoted it ourselves（done by Nan Wang and Nannan Xie).Then we sent the sequences to synthesis,the part can be assembled by gibson assembly,that is what we did.
nifE promoted from Paenibacillus sp. WLY78
NifE(BBa_K1796010) is taken from genomic sequence of Paenibacillus sp.WLY78, we apply promotions on the sequences and synthesised it.
We abtained the sequences from genomes of Paenibacillus sp. WLY78. First, we promoted it ourselvesThen we sent the sequences to synthesis, but unfortunately, striction enzyme cut site was involved after they promoted it again. But the part can be assembled by gibson assembly,that is what we did.
nifN promoted from Paenibacillus sp. WLY78
NifN is taken from genomic sequence of Paenibacillus sp.WLY78, we apply promotions on the sequences and synthesised it.
Function: scaffold gene of nitrogen fixation system
We abtained the sequences from genomes of Paenibacillus sp. WLY78. First, we promoted it ourselves（done by Nan Wang and Nannan Xie).Then we sent the sequences to synthesis, but unfortunately, striction enzyme cut sites were involved(three PstI) after they promoted it again. But the part can be assembled by gibson assembly,that is what we did.
nifX promoted from Paenibacillus sp. WLY78
NifX(BBa_K1796012) is taken from genomic sequence of Paenibacillus sp.WLY78, we apply promotions on the sequences and synthesised it.
Function: encode carrier proteins involved in the delivery of FeMoco to the MoFe protein , participate in FeMoco assembly in a chaperone-like function.
We abtained the sequences from genomes of Paenibacillus sp. WLY78. First, we promoted it ourselves（done by Nan Wang and Nannan Xie).Then we sent the sequences to synthesis, but unfortunately, striction enzyme cut site（one PstI） was involved after they promoted it again. But the part can be assembled by gibson assembly,that is what we did.
Double terminator consisting of BBa_B0010 and BBa_B0012
hesA promoted from Paenibacillus sp. WLY78
It is taken from genomic sequence of Paenibacillus sp.WLY78, we apply promotions on the sequences and synthesised it.
Function has not so far been determined , but Deletion analysis indicates that in addition to the core nif genes, hesA plays an important role in nitrogen fixation and is responsive to the availability of molybdenum. HesA a member of the ThiF-MoeB-HesA family and contains an N-terminal nucleotide binding domain and a C-terminal MoeZ/MoeB-like domain. The ThiF-MoeB-HesA family engage in an ATP-dependent process that activates the C-terminus of partner ubiquitin-like proteins by forming an acyl adenylate complex that facilitates sulfur transfer. First, we promoted it ourselves（done by Nan Wang and Nannan Xie).Then we sent the sequences to synthesis, but unfortunately, striction enzyme cut site was involved after they promoted it again. But we were not informed of the error in promotion.After several times failed expriments, we found the problem.We tackled them over the matter, mistakes were corrected, but the correct genes can't arrive on time,it can only arrive after the deadline. Sequence submit is promoted by the synthesis company, containing a PstI in the gene
nifV promoted from Paenibacillus sp. WLY78
homocitrate synthase，provides the organic moiety in all three nitrogenase cofactors. First, we promoted it ourselves（done by Nan Wang and Nannan Xie).Then we sent the sequences to synthesis, but unfortunately, striction enzyme cut site was involved after they promoted it again. But we were not informed of the error in promotion.After several times failed expriments, we found the problem.We tackled them over the matter, mistakes were corrected, but the correct genes can't arrive on time,it can only arrive after the deadline. Sequence submit is promoted by the synthesis company, containing a PstI in the gene.
We abtained the sequences from genomes of Paenibacillus sp. WLY78. First, we promoted it ourselves.Then we sent the sequences to synthesis, but unfortunately, striction enzyme cut site was involved after they promoted it again. But the part can be assembled by gibson assembly,that is what we did.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Transcriptional terminator consisting of a 64 bp stem-loop.
Conversion of the iGEM parts registry to SBOL2.1