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