BBa_K1975006 1 BBa_K1975006 Red Fluorescent Protein 2016-10-04T11:00:00Z 2016-10-05T07:23:04Z This sequence originally origin from the red sea anemone Discosoma. However, this sequence has been optimized to be used in Bacillus subtilis. This is Red Fluorescent Protein that can be used to tag proteins of interest. It is a fluorescent protein that will show a red color when it is excited at 558nm and it has the highest emission at 583nm. This sequence has been used to tag PhaB to visualize the expression of the protein. If the protein was expressed correctly, the cells would show a red color when excited. The sequence is optimized for use in Bacillus subtilis. false false _2442_ 30862 30862 9 false To secure the optimal expression we choose to make a codon optimization of the genes. This was done through the Codon Optimization tool at IDT's webpage. false Joachim Steen Larsen BBa_K1975007 1 BBa_K1975007 PhaB fused to Red Fluorescent Protein 2016-10-04T11:00:00Z 2016-10-05T08:00:33Z The genetic sequence for PhaB is original from Ralstonia eutropha genomic DNA, but has been optimized to the expression of B. subtilis. The sequence for RFP genomic DNA from Discosoma sp. This sequence has also been optimized for expression in B. subtilis. This is a NADPH-Dependent acetoacetyl-CoA reductase (commonly known as PhaB). This enzyme catalyzes the second step in the production of PHB. The reaction it catalyzes is that it reduces acetoacetyl-CoA to (R)-3-hydroxybutyrate. By combining it with the synthetic gene, LPE it can be used in the synthesis of P(LA-co-3HB). This gene is fused with the Red Fluorescence Protein with a small linker containing the restriction site for NgoMIV, so the tag can be cleaved off. The fused RFP is used to visual the expression of the protein. Hereby, the job will be easier to screen for positive transformants after induction (if such is used as in our case). The sequence for PhaB is taken from the BioBrick Part:BBa_K759005 and is optimized to be expressed in Bacillus subtilis. false false _2442_ 30862 30862 9 false To secure the optimal expression we choose to make a codon optimization of the genes. This was done through the Codon Optimization tool at IDT's webpage. Besides that we looked through the literature to figure our if the PhaB and RFP should be directly attached or if they should have some kind of linker. After looking through different studies that have been working with PhaB in Escherichia coli we came to the conclusion that a linker should not be necessary to secure high expression and activity. We therefore decided to only make a small linker (6 bp) between the gene and the tag. This linker contain a restriction site so it will be possible to digest the construct and then get rid off the fluorescent tag if it is not needed anymore. false Joachim Steen Larsen component2486165 1 BBa_K1975003 component2486166 1 BBa_K1975006 annotation2486166 1 BBa_K1975006 range2486166 1 745 1431 annotation2486165 1 BBa_K1975003 range2486165 1 1 738 BBa_K1975003 1 BBa_K1975003 PhaB - NADPH-dependent acetoacetyl-CoA reductase 2016-06-27T11:00:00Z 2016-06-28T04:13:45Z Genomic DNA Will be coming later... false false _2442_ 30862 30862 9 false Codon optimized for use in B. subtilis false Joachim Steen Larsen BBa_K1975003_sequence 1 atgacacagcgtatagcctatgttacaggtggaatgggggggattggcactgccatctgccagcgcctcgcaaaggatggtttcagagtggttgctggttgcgggccaaacagccctcgtcgtgaaaagtggttggagcaacagaaagcgttgggttttgatttcatagcgtctgagggaaatgtggcagactgggacagcaccaagacagcgttcgataaggtgaaaagtgaagtcggggaggtagacgtactcatcaataacgcgggaataacacgcgatgtggtttttcggaaaatgacccgtgcagattgggacgcagtcatcgacactaatctgacaagccttttcaatgtgactaaacaggtcatcgatggaatggcagatcgcgggtgggggcgtattgtcaacataagttcagtcaatggccagaaaggtcaatttgggcaaacgaattattctacggcaaaagcgggtttacacgggtttacaatggccctggcgcaggaagtcgcaacgaagggagtcacggtgaatactgtttcacctgggtacatagctaccgatatggtaaaagcaatccgtcaagacgttctcgataagatagtcgctactattccagtgaaacggttggggcttccagaagaaatcgcatcaatttgcgcttggttgtcatcagaagagtcagggtttagcaccggtgcagatttctccctgaacgggggccttcatatgggc BBa_K1975007_sequence 1 atgacacagcgtatagcctatgttacaggtggaatgggggggattggcactgccatctgccagcgcctcgcaaaggatggtttcagagtggttgctggttgcgggccaaacagccctcgtcgtgaaaagtggttggagcaacagaaagcgttgggttttgatttcatagcgtctgagggaaatgtggcagactgggacagcaccaagacagcgttcgataaggtgaaaagtgaagtcggggaggtagacgtactcatcaataacgcgggaataacacgcgatgtggtttttcggaaaatgacccgtgcagattgggacgcagtcatcgacactaatctgacaagccttttcaatgtgactaaacaggtcatcgatggaatggcagatcgcgggtgggggcgtattgtcaacataagttcagtcaatggccagaaaggtcaatttgggcaaacgaattattctacggcaaaagcgggtttacacgggtttacaatggccctggcgcaggaagtcgcaacgaagggagtcacggtgaatactgtttcacctgggtacatagctaccgatatggtaaaagcaatccgtcaagacgttctcgataagatagtcgctactattccagtgaaacggttggggcttccagaagaaatcgcatcaatttgcgcttggttgtcatcagaagagtcagggtttagcaccggtgcagatttctccctgaacgggggccttcatatgggctactagatgcgtagctccaagaatgtgatcaaggagtttatgcgctttaaggtgcggatggagggcacagttaacggccacgagtttgaaatcgaaggtgagggagaggggcggccatatgagggccacaatactgttaaattaaaggtgacgaaagggggtcctttaccattcgcctgggatatactcagcccgcaatttcaatacggtagcaaagtgtacgtcaagcatccagcggacattccggattacaaaaaacttagttttccagagggcttcaagtgggagagagtgatgaatttcgaggatggcggcgtcgtgacagtcacccaagactcttctctgcaagacggctgctttatttataaggtgaagtttattggggtaaacttcccatctgacgggccggttatgcagaagaaaacaatggggtgggaggctagcaccgaaagactgtatccgagagatggggtcttaaagggcgagatccacaaagcgttaaaattgaaggacgggggacattacctcgtggagtttaagtctatatatatggctaagaaaccagtgcaacttccaggttactactatgtggactccaaattagatatcacctcacataacgaagattataccattgttgagcaatatgaacgtacagagggaagacaccatttatttcttcaccggtgctaa BBa_K1975006_sequence 1 atgcgtagctccaagaatgtgatcaaggagtttatgcgctttaaggtgcggatggagggcacagttaacggccacgagtttgaaatcgaaggtgagggagaggggcggccatatgagggccacaatactgttaaattaaaggtgacgaaagggggtcctttaccattcgcctgggatatactcagcccgcaatttcaatacggtagcaaagtgtacgtcaagcatccagcggacattccggattacaaaaaacttagttttccagagggcttcaagtgggagagagtgatgaatttcgaggatggcggcgtcgtgacagtcacccaagactcttctctgcaagacggctgctttatttataaggtgaagtttattggggtaaacttcccatctgacgggccggttatgcagaagaaaacaatggggtgggaggctagcaccgaaagactgtatccgagagatggggtcttaaagggcgagatccacaaagcgttaaaattgaaggacgggggacattacctcgtggagtttaagtctatatatatggctaagaaaccagtgcaacttccaggttactactatgtggactccaaattagatatcacctcacataacgaagattataccattgttgagcaatatgaacgtacagagggaagacaccatttatttcttcaccggtgctaa 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