Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Joachim Steen Larsen
Date created: 2016-10-04 11:00:00
Date modified: 2016-10-05 08:00:33
PhaB fused to Red Fluorescent Protein
| Types | DnaRegion |
| Roles | engineered_region Composite |
| Sequences | BBa_K1975007_sequence (Version 1) |
Description
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.Notes
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.Source
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.| Access | Instance | Definition |
| public public | BBa_K1975003 BBa_K1975006 | BBa_K1975003 BBa_K1975006 |
| Sequence Annotation | Location | Component / Role(s) |
| BBa_K1975003 BBa_K1975006 | 1,738 745,1431 | BBa_K1975003 BBa_K1975006 |