Illegal PstI restriction site removed by silent mutations T>A at position 66 and A>T at position 69 using primers listed below (annealing sequences are underlined) ompF_rsr_F CTGAAATCTATAACAAAGATGGCAA ompF_rsr_R CTGCGTTTGCAGTACCTGCT
Amplified from MG1655 (accession NC_000913) chromosome using primers listed below (annealing sequences are underlined) ompF_BB_F TTCGAATTCGCGGCCGCTTCTAGATGATGAAGCGCAATATTC ompF_BB_R CGGCCGCTACTAGTATTATTAGAACTGGTAAACGATACC
Halorhodopsin gene allow chloride ion to be pumped in by the level of light intensity. Halorhodopsin gene is under regulation by constitute T7 promoter to allow protein coding to be maximized, and the bidirectional terminator is added for high efficiency termination of both forward and backward transcription
The double terminator wass tested to stop effectively on the transcription of both forward and backward transcription to make our protein generator be a controlled system
Halorhodopsin comes from part BBa_K559000, which is come from genomic sequence of N.pharaonis (genebank ID:J05199.1)
TonB ferric siderophore transport system, periplasmic binding protein TonB [ Pseudomonas entomophila
This device codify for TonB, a transmembranal Iron channel that appears to transduce energy between the cytoplasmic and the outer membrane in Escherichia coli.
This part encodes the gene GluR0 from Synechocystis. It is a well characterised glutamate-gated K+ membrane channel, which has a considerable degree of structural and functional homology to rat neurone GluR2 AMPA receptors.
From peptide sequence of Synechocystis PCC 6803, codon optimised for E.coli, restriction sites removed, and synthesised de novo.
Halorhodopsin under constitute T7 promoter to regulate the gene transcription to be maximized and no restrict to other regulatory factor
The coding sequence of N.pharaonis J05199.1 is selected from 61nt to 700nt and the T7 promotor placed in front by 3A assembly
Halorhodopsin come from N.pharaonis, gene bank J05199.1
Lactate Permease from Kluyveromyces lactis
Ra??ssa Estrela Curado, Ta??s Sineiro Herig and Gleidson Silva Teixeira
JEN1 from Kluyveromyces lactis is a homologue gene to JEN1 of Saccharomyces cerevisiae. It ecodes a monocarboxylate acids permease shared by lactic and pyruvic acids. Queiros O, Pereira L, Paiva S, Moradas-Ferreira P and Casal M. 2007. Functional analysis of Kluyveromyces lactis carboxylic acids permeases: heterologous expression of KlJEN1 and KlJEN2 genes. Curr Genet 51, 161-169.
This part was amplified by PCR from K. lactis genomic DNA.
ybhGFSR operon encoding an ABC efflux pump
This Halorhodopsin gene allow the photoreceptor protein be expressed, to regulate the chloride ion pumped in by light intensity
The DNA sequence selected from ID J05199.1 of 61nt to 700nt for protein coding
It is from N.pharaonis, gene back ID J05199.1
SrUCPa, a human UCP1 homologue, is a mitocondrial inner membrane protein of skunk cabbage, Symplocarpus renifolius. It facilitates proton leaks across the mitocondrial inner membrane, which confers skunk cabbages the ability to produce heat under low temperature.
coming from genomic DNA of skunk cabbage, Symplocarpus renifolius.
Export of recombinant proteins in Escherichia coli using ABC transporter of Erwinia chrysanthemi
Jung Hoon Ahn from Korea Advanced Institute of Science and Technology
EGF-TliA, EGF-LARD1 fusion proteins were secreted when ABC transporter was present. E. coli harboring the PrtDEF of E. chrysanthemi secreted more fusion proteins than without ABC transporter. In fact, in enzyme activation assay(with Hitachi Spectrophotometer) we observed that although our protein -EGF- has disulfide bonds, it was excreted succesfully. In addition, when compearing with lack of ABC domain, we saw that protein excretion into medium (supernatant) is 10-fold more with ABC transporter-PrtDEF-. PrtDEF functioned well at 37??C, the optimum growth temperature for E. coli
We used whole plasmid in order to excrete a fusion protein with one of two signal tags which are TliA and LARD1
Sequence and Features PrtDEF: ABC transporter of Erwinia chrysanthemi for PrtA EGF: Human Epidermal Growth Factor
Halorhodopsin is an inward-directed light-driven chloride ion pump originating from Halobacterium. It utilizes light to pump chloride ions against chloride concentration difference into cells from the environment. We added a constitutive working and regulatable pLac promoter in the upstream region of the part in order to use it in our design easily.
Please refer to http://partsregistry.org/Part:BBa_K559000:Design for more detail about halorhodopsin. Please refer to http://partsregistry.org/Part:BBa_J04500:Design for more detail about pLac promoter.
Please refer to http://partsregistry.org/Part:BBa_K559000:Design, both of the genes are gathered from the Registry distribution.
ExbB membrane spanning protein in TonB-ExbB-ExbD complex [ Escherichia coli K12 ]
this part codifies for a membran localized protein that transduces energy from the proton motive force to high-affinity receptors in the outer membrane. it is part of the complex composede by a TonB ExbB and ExbD
this part has its original ribosomal binding site
FepA L8T Mutant - Large Diffusion pore for E. coli outer membrane.
FepA is an outer membrane receptor protein for Escherichia coli involved in the uptake of the ferric enterobactin siderophore. This part is a deletion mutant of the wild type FepA, which has been shown to convert the protein into an open diffusion channel, similar to a porin. It allows larger molecules through than normally would be permitted.
The original mutants had an EcoR1 restriction site within their coding sequence. This had to be removed via site directed mutagenesis, using a megaprimer based method.
The mutants were developed by Dr Salete Newton working in Professor P. Klebba's lab at the university of Oklahoma. They were kindly sent to us by Professor Klebba.