BBa_K1840006 1 Kgu-Device Device: TT-kgu-mCherry 2015-09-17T11:00:00Z 2015-09-18T11:39:01Z The double terminator was already registered in the iGEM catalogue. The kgu promotor comes from the genome of Pseudomonas putida. The mCherry is codon optimized for expression in Pseudomonas. This device acts as a detector for glucose (and certain derivatives of it). The double transcription terminator ensures, that only the gene downstream of kgu is influenced by the promotor kgu. The promotor (BBa_K1840000) is contained in the genome of Pseudomonas putida and is situated in front of kgu enzymes (2-ketogluconate epimerase, kinase, transporter and reductase). The repressor PtxS can bind the kgu promotor sequence and thus prevent the transcription of the kgu gene. In case the glucose derivative 2-ketogluconate is present, it binds the promotor and leads to a subsequent conformational change. Hence, the repressor can no longer bind and the gene downstream can be transcribed. In our device, the downstream gene is mCherry, codon optimized for Pseudomonas (BBa_K1840004). Therefore, in the presence of glucose, mCherry is expressed. In various experiments, we could see that this device is in deed working. false false _2266_ 26543 26543 9 false Considerations were made regarding the actual start of the promotor. As promotor sequence we assumed all DNA between the kguE gene and the previous gene. (The previous gene encodes the repressor PtxS.) false Julia Anna Adrian component2473420 1 BBa_K1840001 component2473419 1 BBa_B0014 component2473421 1 BBa_K1840004 annotation2473421 1 BBa_K1840004 range2473421 1 176 889 annotation2473419 1 BBa_B0014 range2473419 1 1 95 annotation2473420 1 BBa_K1840001 range2473420 1 104 169 BBa_B0012 1 BBa_B0012 TE from coliphageT7 2003-01-31T12:00:00Z 2015-08-31T04:07:20Z Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>. Released HQ 2013 Transcription terminator for the <i>E.coli</i> RNA polymerase. false false _1_ 0 24 7 In stock false <P> <P>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).<P> Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator. false Reshma Shetty annotation1687 1 stop range1687 1 34 34 annotation1690 1 polya range1690 1 28 41 annotation1686 1 T7 TE range1686 1 8 27 annotation7020 1 BBa_B0012 range7020 1 1 41 BBa_B0014 1 BBa_B0014 double terminator (B0012-B0011) 2003-07-15T11:00:00Z 2015-08-31T04:07:20Z Released HQ 2013 Double terminator consisting of BBa_B0012 and BBa_B0011 false true _1_ 0 24 7 In stock false true Reshma Shetty component939303 1 BBa_B0012 component939311 1 BBa_B0011 annotation939303 1 BBa_B0012 range939303 1 1 41 annotation939311 1 BBa_B0011 range939311 1 50 95 BBa_B0011 1 BBa_B0011 LuxICDABEG (+/-) 2003-01-31T12:00:00Z 2015-08-31T04:07:20Z Derived from luxICDABEG operon terminator of Vibrio fischeri <genbank>AF170104</genbank>. Released HQ 2013 Bidirectional transcriptional terminator consisting of a 22 bp stem-loop.</p> false false _1_ 0 24 7 In stock false <P> <P>In the naturally-occuring sequence there is a mismatch in the stem of the stem loop. This can be corrected via an A-&gt;G mutation (at position 40 -- sequence coordinate/not MFOLD coordinate). The above sequence does not reflect this mutation (but the MFOLD image does). This terminator's location cannot be found using some inverted repeat detectors like PALINDROME because it is too short and contains a mismatch. This one was found with the help of Tom Knight. It lies between two coding regions that point towards eachother.<P> true Reshma Shetty annotation1683 1 stem_loop range1683 1 13 35 annotation7019 1 BBa_B0011 range7019 1 1 46 BBa_K1840001 1 BBa_K1840001 Kgu promotor from Pseudomonas putida 2015-09-17T11:00:00Z 2015-09-18T01:48:26Z This part was designed after information from the paper by DADDAOUA, A., KRELL, T., ALFONSO, C., MOREL, B. & RAMOS, J.-L. 2010. Compartmentalized Glucose Metabolism in Pseudomonas putida Is Controlled by the PtxS Repressor. Journal of Bacteriology, 192, 4357-4366. This basic part is the promotor in front of four so called kgu genes. They all are associated with 2-ketogluconate (2-ketogluconate epimerase, kinase, transporter and reductase) in Pseudomonas putida. All of them are concerned with the glucose metabolism in P. putida. The kgu promotor is negatively regulated by the repressor called PtxS. The glucose derivative 2-Ketogluconate can bind to it and this leads to subsequent release of PtxS from the promotor. For our project (NTNU Trondheim 2015) we integrated the promotor in front of the mCherry gene. Hence we built a glucose detection device, that expresses mCherry according to the glucose (and therefore 2-Ketogluconate) level in the environment. Note: The promotor gad (BBa_K1840003) is repressed by the same repressor PtxS. See biobrick parts BBa_K1840000 and BBa_K1840002 for other promotors on operons regulated by glucose derivatives. false false _2266_ 26543 26543 9 false The considerations we made regarded the starting and endpoint of the promotor. We only know that it has to be in front of the kgu gene. We assumed it starts directly after the previous gene. This gene codes for PtxS, the repressor. false Julia Anna Adrian BBa_K1840004 1 BBa_K1840004 mCherry for Pseudomonas 2015-09-17T11:00:00Z 2015-09-18T11:02:21Z It comes from the standard mCherry sequence and was condon optimized. The standard fluorescent protein mCherry is not efficiently expressed in Pseudomonas spp. Therefore this biobrick part is a codon optimized mCherry for the expression in Pseudomonas. We used it in our project (NTNU Trondheim 2015) in a device as reporter. Note: See biobrick parts BBa_K1840005 to BBa_K1840008 for the characterization of the devices. false false _2266_ 26543 26543 9 false The expression mechanism in Pseudomonas had to be considered to be able to codon optimize it. false Julia Anna Adrian BBa_K1840001_sequence 1 gcaacggcaggtctgcgcctgctactgccgactaaaaatgaaaccggtttcataaggataaaaaca BBa_B0014_sequence 1 tcacactggctcaccttcgggtgggcctttctgcgtttatatactagagagagaatataaaaagccagattattaatccggcttttttattattt BBa_K1840004_sequence 1 atggtttctaaaggtgaagaagacaacatggctatcatcaaagaatttatgcgtttcaaagttcacatggaaggttctgtgaacggtcacgaatttgaaatcgaaggtgaaggtgaaggtcgtccgtatgaaggcacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtctccgcagttcatgtacggttctaaagcgtatgttaaacacccggctgacatcccggactacctgaaactgtctttcccggaaggtttcaaatgggaacgtgttatgaactttgaagacggtggtgttgttaccgttacccaggactcttctctgcaagacggtgaatttatctacaaagttaaactgcgtggcaccaacttcccgtctgacggtccggttatgcagaaaaaaacgatgggttgggaagcgtcttctgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaacagcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacaaagctaaaaagccggttcaactgccgggtgcttacaacgtgaacatcaaactggacatcacctctcacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactctaccggcggtatggacgaactgtataaatgataa BBa_K1840006_sequence 1 tcacactggctcaccttcgggtgggcctttctgcgtttatatactagagagagaatataaaaagccagattattaatccggcttttttattattttactagaggcaacggcaggtctgcgcctgctactgccgactaaaaatgaaaccggtttcataaggataaaaacatactagatggtttctaaaggtgaagaagacaacatggctatcatcaaagaatttatgcgtttcaaagttcacatggaaggttctgtgaacggtcacgaatttgaaatcgaaggtgaaggtgaaggtcgtccgtatgaaggcacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtctccgcagttcatgtacggttctaaagcgtatgttaaacacccggctgacatcccggactacctgaaactgtctttcccggaaggtttcaaatgggaacgtgttatgaactttgaagacggtggtgttgttaccgttacccaggactcttctctgcaagacggtgaatttatctacaaagttaaactgcgtggcaccaacttcccgtctgacggtccggttatgcagaaaaaaacgatgggttgggaagcgtcttctgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaacagcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacaaagctaaaaagccggttcaactgccgggtgcttacaacgtgaacatcaaactggacatcacctctcacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactctaccggcggtatggacgaactgtataaatgataa BBa_B0011_sequence 1 agagaatataaaaagccagattattaatccggcttttttattattt BBa_B0012_sequence 1 tcacactggctcaccttcgggtgggcctttctgcgtttata 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