BBa_K1355004 1 BBa_K1355004 Mercury ions bioremediating device 2014-10-05T11:00:00Z 2015-05-08T01:10:02Z BBa_K1355001, BBa_K1355000 The biorremediator device is composed by our ???essential biobrick??? (BBa_K1355001) + a MerA gene (BBa_K1355000). In the presence of mercury, The MerR protein will bind to it and MerT, MerP and MerA protein will be expressed and bioremediation will start! The MerP and MerT proteins are responsible for the transport of mercury from the periplasm to the cytoplasm, leading him to the mercury ion reductase (MerA)! The beautiful MerA be responsible for the reduction from Hg2+ in Hg0, which is volatile at room temperature and able to passively leave out the bacteria. false false _1731_ 0 15499 9 In stock true For this construct, we followed these steps: Extraction of plasmid DNA and DNA quantification of bacteria transformed with the Essential Biobrick (BBa_K1355001) and the bacteria transformed with the MerA Biobrick (BBa_E0840); Verifying the electrophoretic profile of the extracted plasmid DNA; Restriction enzyme digestion for Essential Biobrick with SpeI and PstI and for merA Biobrick with PstI and XbaI; Checking the electrophoretic profile of digested samples; Fragment of interest purification from the gel; Ligation the gel purification of merA gene and digested Essential Biobrick; Transformation of ligation in DH5-alpha; Plasmid DNA extraction from bacteria transformed; Check the electrophoretic profile to see results of samples linked (no fragments); Digestion of Essential Biobrick + merA Biobrick with EcoRI and PstI, aiming to analyze the fragment size to be isolated; Checking the electrophoretic profile of the digested sample to obtain results showing that the isolated fragment is the junction of Essential Biobrick +merA Biobrick ; false Luna Barroco de Lacerda, Maria Clara Tavares Astolfi component2415196 1 BBa_K1355001 component2415200 1 BBa_K1355000 annotation2415200 1 BBa_K1355000 range2415200 1 1198 2975 annotation2415196 1 BBa_K1355001 range2415196 1 1 1189 BBa_K1355001 1 BBa_K1355001 Regulation and transport of mercury ions 2014-10-05T11:00:00Z 2015-07-23T04:05:30Z This construction is based on sequences present in the O26-CRL plasmid found in Escherichia coli O26. We added a reverse tryptophan operon terminator to ensure transcription termination of the merR messenger RNA. However we didn???t add transcription terminator after the merP gene, aiming connect this biobrick with others, enabling various functions related to mercury We call this part an ???Essential biobrick??? designed to be the key piece of any genetic construction related to mercury, targeting both a biosensor as a bioaccumulative. This biobrick has bidirectional promoter, the regulator MerR with a terminator reverse from the tryptophan operon and a forward MerP and MerT transporters. false false _1731_ 4206 15499 9 In stock true If is desirable to accumulate mercury, assemble this biobrick with a good RBS, a coding sequence of a peptide having affinity to metal and a terminator! Its function will be regulated by presence of mercury through MerR regulator. The bacteria will carry the mercury to cytoplasm until it is linked to the peptide having affinity to the metal, making a inactive mercury. Is not it amazing? This is the strategy that we use in our project! The so called ???Essential Biobrick??? was connected to biobrick for bioremediation, bioaccumulation and bio-detection! false Luna Barroco de Lacerda annotation2415155 1 MerP range2415155 1 914 1189 annotation2415141 1 MerT range2415141 1 551 900 annotation2415127 1 Bidirecional promoter range2415127 1 486 514 annotation2415126 1 MerR range2415126 1 44 480 annotation2397153 1 Trp Terminator range2397153 1 1 43 annotation2415749 1 RBS range2415749 1 490 496 annotation2415752 1 RBS range2415752 1 537 543 annotation2415134 1 Bidirecional promoter range2415134 1 510 538 annotation2415143 1 RBS range2415143 1 901 907 BBa_K1355000 1 BBa_K1355000 Strong RBS + merA (mercuric ion reductase)+ terminator (BBa_ B0015) 2014-10-05T11:00:00Z 2016-01-13T10:34:49Z MerA gene sequence is found in the O26-CRL plasmid from Escherichia coli O26. We also added strong RBS from protein 10 found in phage 17 and the double terminator (BBa_B0015) to ensure efficient termination of transcription and messenger RNA recognition. The MerA gene is one of the most important gene into bacterial mercury-resistance operon. Transforms mercury Hg2+ in Hg0, which is volatile and moves passively out of the bacteria in gaseous form as shown in Figure above. The reduction of Hg2+ occurs by a NADPH-dependent system. false false _1731_ 4206 15499 9 In stock true We analyzed all Mercury-resistance (Mer) operon genes, in these following strains available at NCBI: (1) Escherichia coli 042, (2) Escherichia coli O26 , (3) Escherichia coli ACN001, (4) Escherichia coli 838C-R1, (5) Escherichia coli APEC-O103, (6) Escherichia coli UMNK88, (7) Escherichia coli AR060302, (8) Escherichia coli PMV-1. It is showed that the mer genes from strains (1) - (2) - (3) - (4) - (5) is conserved among them. However strains (6) - (7) and (8) did not show any similarity. Therefore we used sequences from (1) to (5) due the consensus in E.coli. false Luna Barroco de Lacerda annotation2396893 1 MerA range2396893 1 24 1639 annotation2396890 1 rbs range2396890 1 9 16 annotation2396891 1 BBa_ B0015 range2396891 1 1652 1778 BBa_K1355004_sequence 1 aaagaaagttaaaatgccgccagcggaactggcggctgtgggactaaggcatagctgaccttgccaggcctgcttcgccctgtagtgacgcgatcaacgggcaggaaacattcccctttcgtgcatggcaggcgcacacgagttcagacagcacggtttccatgcgcgccaagtcggccatcttctcgcgcacgtccttgagcttgtgttcggccaggctgctggcctcctcgcagtgggtgccatcgtcgagccgcaacagctcggcaatctcgtccagactgaaccccagccgctgtgccgatttcacgaatttcacccgaaccacgtccgcctccccatagcggcggatgctgccgtaaggcttgtccggttcccgcaacaggcccttgcgctgatagaagcggattgtctccacgttgaccccggccgccttggcaaaaacgccaatggtcaggttttccaaattattttccatatcgcttgactccgtacatgagtacggaagtaaggttacgctatccaatccaaattcaaaagggccaacgtatgtctgaaccacaaaacgggcgcggtgcgctcttcgccggcgggctggccgccattcttgcatcgacctgctgcctggggccgctagtactggtcgccctgggcttctccggtgcttggatcggcaacctgacggtgctggaaccctatcgaccgttgttcatcggcgcggcgctagtggcgctgttcttcgcctggaagcggatttaccggcccgtgcaggcatgcaagccaggtgaggtctgcgcgattccgcaggtgcgcgccacctacaagctgattttctggatcgtggccgtgctggtcctggtcgcgcttggatttccctatgtcgttccatttttctattaaccaggagttcatcatgaagaaactgtttgcctcccttgccctcgccgccgctgttgccccggtgtgggccgctacccagaccgtcacgctagcggttcccggcatgacttgcgccgcctgcccgatcacagtcaagaaagcgctctccaaggtcgaaggcgtgagcaaggtcgatgtgggcttcgagaagcgcgaggccgtcgtcacttttgacgacaccaaggccagcgtacagaagctgaccaaggccaccgcagacgccggctatccgtccagcgtcaagcagtgatactagagaactttaagaaggagatatatccatgcaatcagcggatgtctcctacgccaagggcagcgccaagctcgccattgaggtcggcacgtcacccgacgcgctgacggccgctgtagctggactcggttatcgggccacgctggccgatgccccctcagtttcgacgccgggcggattgctcgacaagatgcgcgatctgctgggcagaaacgacaagacgggtagcagcggcgcattgcatatcgccgtcatcggcagcggcggggccgcgatggcagcggcgctgaaggccgtcgagcaaggcgcacgtgtcacgctgatcgagcgcggcaccatcggcggcacctgcgtcaatgtcggttgtgtgccgtccaagatcatgatccgcgccgcccatatcgcccatctgcgccgggaaagcccgttcgatggcggcatcgccgctaccacgccgaccatccagcgcacggcgctgctggcccagcagcaggcccgcgtcgatgaactgcgccacgccaagtacgaaggcatcttggagggcaatccggcgatcactgtgctgcacggctccgcccgctttaaggacaatcgcaacctgatcgtgcaactcaacgacggcggcgagcgcgtggtggcattcgaccgctgcctgatcgccaccggcgcgagcccggccgtgccgccgattcccggcctgaaagacactccgtactggacttccactgaagcgctggtcagcgagacgattcctaagcgcctggccgtgattggctcatcagtggtggcgctggagctggcgcaggcgttcgcccgactcggagcgaaggtgacgatcctggctcgcagcacgctgttcttccgcgaagacccagctataggcgaagccgtcacggccgcattccgcatggagggcatcgaggtgagggaacacacccaggccagccaggtcgcgtatatcaatggtgaaggggacggcgagttcgtgctcaccacggcgcacggcgaactgcgcgccgacaagctgctggtcgccaccggccgcgcgcccaacacacgcaagctggcactggatgcgacgggcgtcacgctcaccccgcaaggcgctatcgtcatcgaccccggcatgcgtacaagcgtggaacacatctacgccgcaggcgactgcaccgaccagccgcagttcgtctatgtggcggcagcggccggcactcgcgccgcgatcaacatgaccggcggtgacgcggccctgaacctgaccgcgatgccggccgtggtgttcaccgacccgcaagtggcgaccgtaggctacagcgaggcggaagcgcaccatgacggcatcaaaactgatagtcgcacgctaacgctggacaacgtgccgcgcgcgctcgccaacttcgacacgcgcggcttcatcaaactggtggttgaagaaggcagcggacgactgatcggcgtgcaggcagtggccccggaagcgggcgaactgatccagacggccgcactggcgattcgcaaccggatgacggtgcaggaactggccgaccagttgttcccctacctgacgatggtcgaagggttgaagctcgcggcgcagaccttcaacaaggatgtgaagcagctttcctgctgcgccgggtgaggactactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata BBa_K1355000_sequence 1 aactttaagaaggagatatatccatgcaatcagcggatgtctcctacgccaagggcagcgccaagctcgccattgaggtcggcacgtcacccgacgcgctgacggccgctgtagctggactcggttatcgggccacgctggccgatgccccctcagtttcgacgccgggcggattgctcgacaagatgcgcgatctgctgggcagaaacgacaagacgggtagcagcggcgcattgcatatcgccgtcatcggcagcggcggggccgcgatggcagcggcgctgaaggccgtcgagcaaggcgcacgtgtcacgctgatcgagcgcggcaccatcggcggcacctgcgtcaatgtcggttgtgtgccgtccaagatcatgatccgcgccgcccatatcgcccatctgcgccgggaaagcccgttcgatggcggcatcgccgctaccacgccgaccatccagcgcacggcgctgctggcccagcagcaggcccgcgtcgatgaactgcgccacgccaagtacgaaggcatcttggagggcaatccggcgatcactgtgctgcacggctccgcccgctttaaggacaatcgcaacctgatcgtgcaactcaacgacggcggcgagcgcgtggtggcattcgaccgctgcctgatcgccaccggcgcgagcccggccgtgccgccgattcccggcctgaaagacactccgtactggacttccactgaagcgctggtcagcgagacgattcctaagcgcctggccgtgattggctcatcagtggtggcgctggagctggcgcaggcgttcgcccgactcggagcgaaggtgacgatcctggctcgcagcacgctgttcttccgcgaagacccagctataggcgaagccgtcacggccgcattccgcatggagggcatcgaggtgagggaacacacccaggccagccaggtcgcgtatatcaatggtgaaggggacggcgagttcgtgctcaccacggcgcacggcgaactgcgcgccgacaagctgctggtcgccaccggccgcgcgcccaacacacgcaagctggcactggatgcgacgggcgtcacgctcaccccgcaaggcgctatcgtcatcgaccccggcatgcgtacaagcgtggaacacatctacgccgcaggcgactgcaccgaccagccgcagttcgtctatgtggcggcagcggccggcactcgcgccgcgatcaacatgaccggcggtgacgcggccctgaacctgaccgcgatgccggccgtggtgttcaccgacccgcaagtggcgaccgtaggctacagcgaggcggaagcgcaccatgacggcatcaaaactgatagtcgcacgctaacgctggacaacgtgccgcgcgcgctcgccaacttcgacacgcgcggcttcatcaaactggtggttgaagaaggcagcggacgactgatcggcgtgcaggcagtggccccggaagcgggcgaactgatccagacggccgcactggcgattcgcaaccggatgacggtgcaggaactggccgaccagttgttcccctacctgacgatggtcgaagggttgaagctcgcggcgcagaccttcaacaaggatgtgaagcagctttcctgctgcgccgggtgaggactactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata BBa_K1355001_sequence 1 aaagaaagttaaaatgccgccagcggaactggcggctgtgggactaaggcatagctgaccttgccaggcctgcttcgccctgtagtgacgcgatcaacgggcaggaaacattcccctttcgtgcatggcaggcgcacacgagttcagacagcacggtttccatgcgcgccaagtcggccatcttctcgcgcacgtccttgagcttgtgttcggccaggctgctggcctcctcgcagtgggtgccatcgtcgagccgcaacagctcggcaatctcgtccagactgaaccccagccgctgtgccgatttcacgaatttcacccgaaccacgtccgcctccccatagcggcggatgctgccgtaaggcttgtccggttcccgcaacaggcccttgcgctgatagaagcggattgtctccacgttgaccccggccgccttggcaaaaacgccaatggtcaggttttccaaattattttccatatcgcttgactccgtacatgagtacggaagtaaggttacgctatccaatccaaattcaaaagggccaacgtatgtctgaaccacaaaacgggcgcggtgcgctcttcgccggcgggctggccgccattcttgcatcgacctgctgcctggggccgctagtactggtcgccctgggcttctccggtgcttggatcggcaacctgacggtgctggaaccctatcgaccgttgttcatcggcgcggcgctagtggcgctgttcttcgcctggaagcggatttaccggcccgtgcaggcatgcaagccaggtgaggtctgcgcgattccgcaggtgcgcgccacctacaagctgattttctggatcgtggccgtgctggtcctggtcgcgcttggatttccctatgtcgttccatttttctattaaccaggagttcatcatgaagaaactgtttgcctcccttgccctcgccgccgctgttgccccggtgtgggccgctacccagaccgtcacgctagcggttcccggcatgacttgcgccgcctgcccgatcacagtcaagaaagcgctctccaaggtcgaaggcgtgagcaaggtcgatgtgggcttcgagaagcgcgaggccgtcgtcacttttgacgacaccaaggccagcgtacagaagctgaccaaggccaccgcagacgccggctatccgtccagcgtcaagcagtga 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