BBa_K535004 1 BBa_K535004 NifH promoter -> Rhizobium etli 2011-09-24T11:00:00Z 2015-05-08T01:12:36Z This is the upstream regulatory sequence of the nifHa gene in Rhizobium etli, which encodes a nitrogenase reductase. The R. etli nifHa gene is located on the symbiotic plasmid p42d, which is a circular molecule of 371, 255 bp that has the repABC type of replicator. There are three copies of the nitrogenase reductase genes in this plasmid: nifHa, nifHb and nifHc. The nifHa and nifHb copies are transcriptionally coupled to nitrogenase structural genes, forming the nifHDK operons [2]. The nifHc copy is linked to a truncated nifD homolog. A remarkable difference among them is the absence of canonical NifA binding sites upstream nifHc while a canonical binding site is located 200 bp upstream of nifHa and nifHb. This is the promoter region of one of the three nitrogenase reductase gene copies in Rhizobium etli, nitrogenase reductase a (nifHa). This region comprises a σ54-dependent promoter located between position -24 and -12 relative to the transcription start site, as well as a binding site for NifA, a positive transcriptional regulator [1]. NifA is a member of the enhancer-binding protein (EBP) family of transcriptional regulators that activate gene expression in concert with RNA polymerase containing the specialized σ54 sigma factor (RpoN) to allow the polymerase core to recognize the -24/-12 promoter [3] in response to oxygen and/or fixed nitrogen levels. The binding site for NifA is located ~200 bp upstream of the transcription start site [2]. Activation of gene expression by NifA (along with the specialized sigma factor σ54) occurs only at low oxygen concentrations [2]. In R. etli, the nifA gene is located on the symbiotic plasmid [4]. This promoter region, in addition to other similar promoter regions ensure the expression of the nitrogen fixation apparatus during symbiosis of the bacteroid with its legume host [3]. We decided to use this promoter region in our project to promote the transcription of gene constructions that, according to our design, need to be expressed in low oxygen conditions in R. etli. The genes regulated by this promoter region will generally be active in the bacteroid state of R. etli because there are low concentrations of oxygen in the nodule???s environment. false false _701_ 0 8698 9 It's complicated false This regulatory sequence was amplified by PCR using the genome of R. etli strain CFN42 as template DNA. The oligos used to amplify the promoter region from the mentioned genome are the following: Upper primer: this primer contains the prefix sequence along with the first 25 nt of the promoter region. CGGAATTCGCGGCCGCTTCTAGAGCGACACCGTCTGTCGGCTTTGTCTG Lower primer: this primer contains the suffix sequence along with the last 24 nt of the promoter region (they do not include the RBS we used in our design). AACTGCAGCGGCCGCTACTAGTATCTTTCGTTCCATCCATCGCTGAG *and has no modifications according to the reported sequence by Emmanuel Salazar, 2010. false iGEM11_UNAM-Genomics_ Mexico annotation2144856 1 -24/-12 box range2144856 1 125 141 annotation2144855 1 Enhancer binding site range2144855 1 19 35 annotation2144854 1 nifH promoter range2144854 1 1 203 BBa_K535003 1 BBa_K535003 FeOx -> Clostridium acetobutylicum???s ferredoxin 2011-09-24T11:00:00Z 2015-05-08T01:12:36Z This sequence was copied from the ferredoxin gene encoded in C. acetobutylicum???s genome. Ferredoxins are small soluble proteins that contain iron-sulfur clusters and that usually mediate electron transfer in a range of metabolic reactions. Their iron-sulfur clusters contain iron and sulfur atoms organized, the iron atoms can change in the oxidation state (+2 or +3) and in that way accept or discharge electrons. Thus, ferredoxins act as electron transfer agents in biological redox reactions. In this case we use this Clostridium acetobutylicum???s ferredoxin to transfer electrons to a linked hydrogenase from the same organism. PFOR should oxidize pyruvate to acetyl-CoA and then reduce the ferrodoxin so it can transfer the electron. false false _701_ 0 8698 9 Not in stock false Some codons of the original Clostridium acetobutylicum FeOx sequence have been changed for synonimous ones according to the Codon Adaptation Index (CAI) procedure with respect to Rhizobium etli CFN42 codon usage in order to optimize its expression and to optimize R. etli CFN42???s (where we will express this gene) fitness as well. The Codon adaptation Index indicates how similar the Codon Usage (CU) in a coding sequence (CDS) is to that of highly/constitutively expressed genes. It is not a cause of high gene expression, but it is necessary to optimize resource usage. To optimize a sequence according to the CAI procedure we first obtained relative adaptiveness (w) for each codon (1.- most frequent codon. 0.- non-existent codon) in R. etli and then we substitute codons in target CDS for all synonymous codons with greatest w. In our final construction (part ####) we coupled this sequence with the N-terminus hydA gene (part ####) using a flexible glycine/serine-rich linker of 14 aminoacid long in order to make the electron transfer more efficient. At the end we added a poly-His region so we can make an immuno-assay to verify that the whole construction is being exported to the periplasm, this tag is flanked by two AatII restriction sites so we can split it out if needed. We also added a double TAA terminator. The whole construction is regulated by the NifH promoter region (part ###) so it will be transcribed under microaerobic conditions. This part was synthesized. false iGEM11_UNAM-Genomics_ Mexico annotation2139560 1 linker range2139560 1 1 42 annotation2139563 1 double TAA stop range2139563 1 241 246 annotation2139561 1 FeOx range2139561 1 43 210 annotation2139562 1 poly His tag range2139562 1 211 240 BBa_K535002 1 BBa_K535002 HydA (hydrogenase I)-> Clostridium acetobutylicum ATCC 824 2011-09-24T11:00:00Z 2015-05-08T01:12:36Z This sequence belongs to the Clostridium acetobutylicum ATCC 824 hydA hydrogenase gene which is in the chromosome of this organism. It has been modified, please refer to the ???design notes??? section for more details. Hydrogenases are a class of metalloenzymes that catalyze the reversible reduction of protons to molecular hydrogen 2H+ + 2e− ↔ H2 with an equilibrium constant that is dependent on the reducing potential of electrons carried by their redox partner. These metalloenzymes (containing metallo-catalytic clusters) are subdivided into two classes depending of the two metal atoms that are present at their active center: either a Ni and a Fe atom in the [NiFe]hydrogenases, or two Fe atoms in the [FeFe]hydrogenases. These two forms are phylogenetically distinct, which suggests that hydrogenase function is the result of convergent evolution. [NiFe] hydrogenases are found across a variety of organisms, [FeFe] hydrogenases are typically restricted to algal species and to a few anaerobic prokaryotes, such as clostridia and sulfate reducers, but are excluded from all cyanobacteria examined to date. Although [NiFe] and [Fe] hydrogenases are genetically unrelated, similarities between the proteins do exist. First, the active sites of both enzymes contain CO and CN ligands, and, second, each active site contains a binuclear metal center. The hydrogenase presented here is a [FeFe] hydrogenase from Clostridium acetobutylicum ATCC 824 which is a Gram-positive bacteria that belongs to the Firmicutes division and that is an obligate anaerobe capable of produce endospores. We choose this hydrogenase because [FeFe]-hydrogenases, such as those from Clostridium species contain several ???ferredoxin-like??? domains. It is speculated that these domains arose through ancestral gene fusions, enhancing hydrogenase interaction with other ferredoxins, and providing an electron transport channel towards the hydrogenase active site. Because ferredoxin proteins may carry electrons with reducing potentials closer to that of the H2∕H+ pair (−420 mV), [FeFe]-hydrogenases thermodynamically favor hydrogen production relative to [NiFe] hydrogenases, which are generally coupled to NAD(P)H, with a reducing potential of -320 mV and are frequently regarded as predominantly H2 uptake enzymes. [Fe] hydrogenase catalytic site is known as the H-cluster and consists of a [4Fe4S] cluster connected through a bridging cysteinyl ligand to a binuclear [2Fe] center. In addition to binding CO and CN, the iron atoms of the [2Fe] center coordinate a bridging organic group thought to be a di(thio-methyl)amine moiety. The H-clusters of [Fe] hydrogenases are easily oxidized and are located in the interior of the protein structure. These sites are connected to the surface by a hydrophobic channel that facilitates H2 diffusion. Because of the complexity of the [Fe] hydrogenase H-cluster assembly, the active hydrogenase expression require at least three accessory proteins called the HydE, HydF, and HydG maturases, like the [FeFe] hydrogenases, HydE, HydF, and HydG also require ISCs (iron-sulfur clusters). The reduced nature of the H-cluster and accessory iron-sulfur clusters (ISCs) makes the Hydrogenase and the maturases susceptible to damage by O2 oxidation. false false _701_ 0 8698 9 Not in stock false Some codons of the original Clostridium acetobutylicum ATCC 824 hydA sequence have been changed for synonimous ones according to the Codon Adaptation Index (CAI) procedure with respect to Rhizobium etli CFN42 codon usage in order to optimize its expression and to optimize R. etli CFN42???s (where we will express this gene) fitness as well. The Codon adaptation Index indicates how similar the Codon Usage (CU) in a coding sequence (CDS) is to that of highly/constitutively expressed genes. It is not a cause of high gene expression, but it is necessary to optimize resource usage. To optimize a sequence according to the CAI procedure we first obtained relative adaptiveness (w) for each codon (1.- most frequent codon. 0.- non-existent codon) in R. etli and then we substitute codons in target CDS with all synonymous codons with greatest w. The sequence that we use is linked to the FeOx gene, at the end it has two TAA stop codons. Please refer to the composite part BBa_K535###. Unwanted restriction sites had been changed for synonimous codons. A NifH promoter from Rhizobium etli CFN42 has been added to regulate the expression of this construction because we want it to be expressed under the same conditions that the genes related to nitrogen fixation are expressed. This sequence was synthesized . false iGEM11_UNAM-Genomics_ Mexico annotation2139546 1 HydA range2139546 1 139 1884 annotation2139545 1 Periplasm export tag range2139545 1 1 138 BBa_K535005 1 BBa_K535005 HydA1 - FeOx construction (Hydrogenase-ferredoxin fussion) 2011-09-24T11:00:00Z 2015-05-08T01:12:36Z Both genes are part of Clostridium acetobutylicum ATCC 824 chromosome, some modifications to the natural sequence were made, for more details check out the ???design notes??? section. Ferredoxin NCBI accession number NP_346944; Hydrogenase NCBI accession number AAB03723. The hydrogenase HydA1 requires electrons to reduce protons and form hydrogen gas molecules. The ferredoxin is the protein that mediates the electron transfer between the an electron transfer donnor and a hydrogenase. The ferredoxin and the hydrogenase need to physically interact for the circuit to function, actually their interaction surface has been extensively modeled in silico, with evidence that this interaction has a strong electrostatic component. The putative ferredoxin-binding region is on the N-terminal domain of the hydrogenase, which includes all of the F-clusters that transfer electrons from the surface to the active-site H-cluster. Experiments had been done in order to improve hydrogen production through physically linking the hydrogenase and ferredoxin to increase the chance of binding and electron transfer between the desired partners. In vivo (in E. coli) activity of the fusion proteins when coexpressed with the PFOR from Desulfovibrio africanus depended on linker length as well as overall configuration. The highest improvement was seen when the ferredoxin is fused to the hydrogenase N-terminus with a flexible glycine/serine-rich linker of 14 aminoacid long which led to a 3-5 fold increase in hydrogen output from the scaffolded circuit. We express this hydrogenase-ferredoxintranslational fusion from Clostridium acetobutylicum ATCC 824 in Rhizobium etli CFN42 with a Desulfovibrio africanus PFOR (Pyruvate Ferredoxin Oxidoreductase) to make the entire circuit. This construction is regulated by a nifH promoter from Rhizobium etli CFN42 which promotes transcription under the conditions that favor nitrogen fixation which includes a microanaerobic environment, necessary for HydA correct function. false false _701_ 0 8698 9 Not in stock false Some codons of the original Clostridium acetobutylicum ATCC 824 HydA1 and FeOx sequences have been changed for synonimous ones according to the Codon Adaptation Index (CAI) procedure in order to optimize its expression and to optimize Rhizobium etli CFN42 fitness as well. The CAI indicates how similar the Codon Usage (CU) in a coding sequence (CDS) is to that of highly/constitutively expressed genes. It is not a cause of high gene expression, but it is necessary to optimize resource usage. To optimize a sequence according to the CAI procedure we first obtained relative adaptiveness (w) for each codon (1.- most frequent codon. 0.- non-existent codon) in R. etli and then we substitute codons in target CDS with all synonymous codons with greatest w. The fusion between HydA1 and FeOx is mediated by a 14aa long glycine/serine rich linker. The whole construction transcription is mediated by a Rhizobium etli CFN42 nifH promoter which is active under the nitrogen fixation conditions which include low oxygen concentration. We added at the end a double TAA terminator. A NifH promoter from Rhizobium etli CFN42 has been added to regulate the expression of this construction because we want it to be expressed under the same conditions that the genes related to nitrogen fixation are expressed. Unwanted restriction sites had been changed for synonimous codons. A 6 aminoacid long polyhistidine tag has been added at the C-terminus. This tag is flanked by two HindIII restriction sites. false iGEM11_UNAM-Genomics_ Mexico component2219966 1 BBa_K535002 component2219963 1 BBa_K535004 component2219971 1 BBa_K535003 annotation2219966 1 BBa_K535002 range2219966 1 210 2093 annotation2219963 1 BBa_K535004 range2219963 1 1 203 annotation2219971 1 BBa_K535003 range2219971 1 2102 2347 BBa_K535003_sequence 1 ggcggcggctcgggcggcggcggctcgggcggcggcggctcgatggcctataagatcaccgacgcctgcgtctcgtgcggctcgtgcgcctcggagtgcccggtctcggccatctcgcagggcgacacccagttcgtcatcgacgccgacacctgcatcgagtgcggcaactgcgccaacgtctgcccggtcggcgccccggtccaggagaagcttcatcatcatcatcatcataagctttaataa BBa_K535004_sequence 1 cgacaccgtctgtcggctttgtctgatcggcgacattaggtttgtttggcagtttcctgtggtggttcggagtaactttctgaaacccaacaaaaggatctttcctttggctcgatcggcccacatggcacgggttttgaagattgccatgcgaggcggcgcgagctgcctgccttttactcagcgatggatggaacgaaaga BBa_K535005_sequence 1 cgacaccgtctgtcggctttgtctgatcggcgacattaggtttgtttggcagtttcctgtggtggttcggagtaactttctgaaacccaacaaaaggatctttcctttggctcgatcggcccacatggcacgggttttgaagattgccatgcgaggcggcgcgagctgcctgccttttactcagcgatggatggaacgaaagatactagatgccgtcgtaccgccccccgaagatcgcctcgtcggagatcaccccgcgccaggtctatctgcgccgccgcgagttcctgggcgccgccaccctgggcgccatggccctgtatggcgccggcaaggcctcggcccatatgaagaccatcatcctgaacggcaacgaggtccataccgacaaggacatcaccatcctggagctggcccgcgagaacaacgtcgacatcccgaccctgtgcttcctgaaggactgcggcaacttcggcaagtgcggcgtctgcatggtcgaggtcgagggcaagggcttccgcgccgcctgcgtcgccaaggtcgaggacggcatggtcatcaacaccgagtcggacgaggtcaaggagcgcatcaagaagcgcgtctcgatgctgctggacaagcatgagttcaagtgcggccagtgctcgcgccgcgagaactgcgagttcctgaagctggtcatcaagaccaaggccaaggcctcgaagccgttcctgccggaggacaaggacgccctggtcgacaaccgctcgaaggccatcgtcatcgaccgctcgaagtgcgtcctgtgcggccgctgcgtcgccgcctgcaagcagcatacctcgacctgctcgatccagttcatcaagaaggacggccagcgcgccgtcggcaccgtcgacgacgtctgcctggacgactcgacctgcctgctgtgcggccagtgcgtcatcgcctgcccggtcgccgccctgaaggagaagtcgcatatcgagaaggtccaggaggccctgaacgacccgaagaagcatgtcatcgtcgccatggccccgtcggtccgcaccgccatgggcgagctgttcaagatgggctatggcaaggacgtcaccggcaagctgtataccgccctgcgcatgctgggcttcgacaaggtcttcgacatcaacttcggcgccgacatgaccatcatggaggaggccaccgagctgctgggccgcgtcaagaacaacggcccgttcccgatgttcacctcgtgctgcccggcctgggtccgcctggcccagaactatcatccggagctgctggacaacctgtcgtcggccaagtcgccgcagcagatcttcggcaccgcctcgaagacctattatccgtcgatctcgggcatcgccccggaggacgtctataccgtcaccatcatgccgtgcaacgacaagaagtatgaggccgacatcccgttcatggagaccaactcgctgcgcgacatcgacgcctcgctgaccacccgcgagctggccaagatgatcaaggacgccaagatcaagttcgccgacctggaggacggcgaggtcgacccggccatgggcacctattcgggcgccggcgccatcttcggcgccaccggcggcgtcatggaggccgccatccgctcggccaaggacttcgccgagaacaaggagctggagaacgtcgactataccgaggtccgcggcttcaagggcatcaaggaggccgaggtcgagatcgccggcaacaagctgaacgtcgccgtcatcaacggcgcctcgaacttcttcgagttcatgaagtcgggcaagatgaacgagaagcagtatcatttcatcgaggtcatggcctgcccgggcggctgcatcaacggcggcggccagccgcatgtcaacgccctggaccgcgagaacgtcgactatcgcaagctgcgcgcctcggtcctgtataaccaggacaagaacgtcctgtcgaagcgcaagtcgcatgacaacccggccatcatcaagatgtatgactcgtatttcggcaagccgggcgagggcctggcccataagctgctgcatgtcaagtataccaaggacaagaacgtctcgaagcatgagtactagagggcggcggctcgggcggcggcggctcgggcggcggcggctcgatggcctataagatcaccgacgcctgcgtctcgtgcggctcgtgcgcctcggagtgcccggtctcggccatctcgcagggcgacacccagttcgtcatcgacgccgacacctgcatcgagtgcggcaactgcgccaacgtctgcccggtcggcgccccggtccaggagaagcttcatcatcatcatcatcataagctttaataa BBa_K535002_sequence 1 atgccgtcgtaccgccccccgaagatcgcctcgtcggagatcaccccgcgccaggtctatctgcgccgccgcgagttcctgggcgccgccaccctgggcgccatggccctgtatggcgccggcaaggcctcggcccatatgaagaccatcatcctgaacggcaacgaggtccataccgacaaggacatcaccatcctggagctggcccgcgagaacaacgtcgacatcccgaccctgtgcttcctgaaggactgcggcaacttcggcaagtgcggcgtctgcatggtcgaggtcgagggcaagggcttccgcgccgcctgcgtcgccaaggtcgaggacggcatggtcatcaacaccgagtcggacgaggtcaaggagcgcatcaagaagcgcgtctcgatgctgctggacaagcatgagttcaagtgcggccagtgctcgcgccgcgagaactgcgagttcctgaagctggtcatcaagaccaaggccaaggcctcgaagccgttcctgccggaggacaaggacgccctggtcgacaaccgctcgaaggccatcgtcatcgaccgctcgaagtgcgtcctgtgcggccgctgcgtcgccgcctgcaagcagcatacctcgacctgctcgatccagttcatcaagaaggacggccagcgcgccgtcggcaccgtcgacgacgtctgcctggacgactcgacctgcctgctgtgcggccagtgcgtcatcgcctgcccggtcgccgccctgaaggagaagtcgcatatcgagaaggtccaggaggccctgaacgacccgaagaagcatgtcatcgtcgccatggccccgtcggtccgcaccgccatgggcgagctgttcaagatgggctatggcaaggacgtcaccggcaagctgtataccgccctgcgcatgctgggcttcgacaaggtcttcgacatcaacttcggcgccgacatgaccatcatggaggaggccaccgagctgctgggccgcgtcaagaacaacggcccgttcccgatgttcacctcgtgctgcccggcctgggtccgcctggcccagaactatcatccggagctgctggacaacctgtcgtcggccaagtcgccgcagcagatcttcggcaccgcctcgaagacctattatccgtcgatctcgggcatcgccccggaggacgtctataccgtcaccatcatgccgtgcaacgacaagaagtatgaggccgacatcccgttcatggagaccaactcgctgcgcgacatcgacgcctcgctgaccacccgcgagctggccaagatgatcaaggacgccaagatcaagttcgccgacctggaggacggcgaggtcgacccggccatgggcacctattcgggcgccggcgccatcttcggcgccaccggcggcgtcatggaggccgccatccgctcggccaaggacttcgccgagaacaaggagctggagaacgtcgactataccgaggtccgcggcttcaagggcatcaaggaggccgaggtcgagatcgccggcaacaagctgaacgtcgccgtcatcaacggcgcctcgaacttcttcgagttcatgaagtcgggcaagatgaacgagaagcagtatcatttcatcgaggtcatggcctgcccgggcggctgcatcaacggcggcggccagccgcatgtcaacgccctggaccgcgagaacgtcgactatcgcaagctgcgcgcctcggtcctgtataaccaggacaagaacgtcctgtcgaagcgcaagtcgcatgacaacccggccatcatcaagatgtatgactcgtatttcggcaagccgggcgagggcctggcccataagctgctgcatgtcaagtataccaaggacaagaacgtctcgaagcatgag igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 Chris J. Myers James Alastair McLaughlin 2017-03-06T15:00:00.000Z