This part includes optimized mhbM using Codon Optimization Tool of IDT and a designed rbs using RBS Calculator.
PbaC comes from Sphingobium wenxiniae JZ-1, and it exists on the Scaffold 12, instead of the vector of pbaA1A2B.Sphingobium wenxiniae JZ-1 utilizes a wide range of pyrethroids and their metabolic product, 3-phenoxybenzoate, as sources of carbon and energy. GenBank: KJ009325.1 reference: A Novel Angular Dioxygenase Gene Cluster Encoding 3-Phenoxybenzoate 1=,2=-Dioxygenase in Sphingobium wenxiniae JZ-1, Chenghong Wang, Qing Chen, Rui Wang, Chao Shi, Xin Yan, Jian He, Qing Hong, Shunpeng Li Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, China.
PbaC is a 44-kDa protein that consists of 408 amino acids and contains a consensus motif for a flavin adenine dinucleotide-binding (ADP-binding) site (GXGX2GX3A X6G)。Existing in Sphingobium wenxiniae JZ-1, it???s a glutathione reductase (GR)-type reductase gene. Though pbaC is the important gene to degrade 3-PBA as the first step, the transcriptional level of pbaC was far less than that of other genes like pbaA1A2B since it is not located in the immediate vicinity of pbaA1A2B. However, with the optimization of our experiment, the efficience of degradation on 3-pba has been improved.
we have used diffrerent promotors and RBS to test the degrading efficience of pbaC. And we design the sequence as the part submission requests.
This is a compostie part including a putative 3-hydroxybenzoate transporter gene(mhbT) from Klebsiella pneumoniae strain M5a1.
This composite part includes a constitutive promoter (BBa_J23100), a RBS(BBa_B0034), a putative 3-hydroxybenzoate transporter gene(mhbT) and the double terminator (BBa_B0015). The gene MhbT is a transport protein which is located on the cytomembrane. It can transport the 3-HBA into the cell.By sequence comparison. This composite part can be used in degradation system of 3-HBA, and can also be used in induction system whose derivant is 3-HBA.
Uses J23100, a strong Anderson Promoter, to produce high levels of expression of mhbT.
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Double terminator consisting of BBa_B0010 and BBa_B0012
Double terminator consisting of BBa_B0012 and BBa_B0011
PbaC comes from Sphingobium wenxiniae JZ-1, and it exists on the Scaffold 12, instead of the vector of pbaA1A2B.Sphingobium wenxiniae JZ-1 utilizes a wide range of pyrethroids and their metabolic product, 3-phenoxybenzoate, as sources of carbon and energy.
It???s a codon-optimized gene of pbaC(BBa_K2088003), and it can be used with provided standard restriction site (EcoRI,XbaI,SpeI,PstI) without destructing the functional region of pbaC. PbaC is the phenoxybenzoic acid dioxygenase reductase on Sphingobium wenxiniae's genomic DNA and it codes a 44-kDa protein that consists of 408 amino acids and contains a consensus motif for a flavin adenine dinucleotide-binding (ADP-binding) site (GXGX2GX3AX6G)。Existing in Sphingobium wenxiniae JZ-1, it???s a glutathione reductase (GR)-type reductase gene. Though pbaC is the important gene to degrade 3-PBA as the first step, the transcriptional level of pbaC was far less than that of other genes like pbaA1A2B since it is not located in the immediate vicinity of pbaA1A2B. However, with the optimization of our experiment, the efficience of degradation on 3-pba has been improved.
We optimaized the codon region of pbaC for a more convient use if other teams want to use it.
MhbR: a protein that combines the 3HBA to induce the promoter Mhbpt.
The gene of MhbR can be gotten from the bacteria M5a1 using PCR.The sequence of the gene comes from GenBank https://www.ncbi.nlm.nih.gov/nuccore/EU165545.1
MhbR is a protein that can combine the 3HBA to induce the promoter Mhbpt.MhbR can be used in the induction system and the system will be induced when both of the MhbR and the 3-HBA exist. It can also be used as a section of the detection device when we need to degrade the 3-HBA.
MhbT is responsible for the uptake of 3-hydroxybenzoate
We get the part from the bacteria M5a1, and the sequence of the gene was found through Genbank https://www.ncbi.nlm.nih.gov/nuccore/AY648560.1
MhbT is a transport protein which is located on the cytomembrane. It can transport the 3-HBA into the cell.By sequence comparison, MhbT exhibited 42% identity with the 4-hydroxybenzoate and protocatechuate transporter PcaK from P.putida, 27% identity with the benzoate transporter BenK from Acinetobacter sp. Using the MhbT-GFP fusion proteins , we can find that MhbT is located to the periphery of cells. It can be used in degradation system of 3-HBA, and can also be used in induction system whose derivant is 3-HBA.
Derived from luxICDABEG operon terminator of Vibrio fischeri <genbank>AF170104</genbank>.
Bidirectional transcriptional terminator consisting of a 22 bp stem-loop.
In the naturally-occuring sequence there is a mismatch in the stem of the stem loop. This can be corrected via an A->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.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
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). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Transcriptional terminator consisting of a 64 bp stem-loop.
Conversion of the iGEM parts registry to SBOL2.1