Oxygen-dependent EGF synthesizing with GFP reporter
EGF will be activated with oxygen presence using tetR inverter mechanism. Therefore,with the absence of oxygen tetR will be synthesized so that EGF and GFP synthesize will be stopped.
the Epidermal Growth Factor(EGF) has a big role in wound healing.Moreover, it has also a function to start the formation of keratin and to stimulate cell division. It can also increase the amount of nucleic acids.And, it causes the storage of glikozamin and glikozaminoglikan.In structural perspective, it is composed of 53 aminoacids and has three disulfide bond.Although these positive effect of EGF, with the abundance, it can show ,for example, a trigger effect of tumor cells formation. Another side effect is to start the formation of vein.As a result, it may show a trigger for cancer unless unregulated and too much synthesize..
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
Built by Josh as an intermediate in screening plasmid construction
Thermostable lipase (TliA) of Pseudomonas fluorescens SIK W1
The thermostable lipase (TliA) of P. fluorescens SIK W1 is comprised of 476 amino acids and has the characteristic C-terminal signal sequence recognized by the ABC transporter.
TliA has four glycinerich repeats (GGXGXD) in its C-terminus, which appear in many ABC transporter-secreted proteins.Export of fusion proteins with the whole TliA through the ABC transporter was evident on the basis of lipase enzymatic activity. Upon supplementation of E. coli with ABC transporter, EGF-TliA was excreted into the culture supernatant.Whole TliA were attached to C-termini of model proteins and enabled the export of the model proteins such as GFP and EGF which has 3 disulfide bonds in E. coli supplemented with ABC transporter. Activity domain (residues 1???268) and secretion/chaperon domain (residues 279???476). In our experiment, we observed that TliA fused proteins were excreted to supernatant culture succesfully by detecting lipase activity with tributyrin and spectrophotometric detection with the substrate p-nitrophenyl phosphate at 420 nm. At the experiments, Tlia fused proteins were excreted ten-fold more with ABC transporter PrtDEF of Erwinia chrysanthemi.
The sequence was optimized for E.coli with Biobrick restriction sites
Jung Hoon Ahn from Korea Advanced Institute of Science and Technology
EGF is synthesized in human and some mammalians. Our EGF was synthesized synthetically at GENEART
Epidermal growth factor (EGF) is a single chain, 53 aminoacid-residue polypeptide (molecular weight 6045) and three intramolecular disulfide bonds. EGF is a low-molecular-weight polypeptide first purified from the mouse submandibular gland, but since then found in many human tissues including submandibular gland, parotid gland. EGF results in cellular proliferation, differentiation, and survival. EGF stimulates the growth of skin and corneal epithelium in vivo and in organ cultures. Epidermal growth factor (EGF) stimulates the growth of keratinocytes in vivo, and therefore plays an important role in the process of wound healing that depends on mitosis and migration of keratinocytes. Rhinewald and Green showed, in vitro that in the presence of growth factors , higher percentage of cells leave the resting state, enter and remain in the mitotic cycle. Mitogenic effect of EGF requires continuous exposure of target cells to EGF for a minimum 6-12 hours. The stimulation of wound healing by EGF has been confirmed as growth of granulation tissue in sponge implants used as inductive matrices. They observed, in rats, dose-dependent effect of EGF on granulation tissue formation as increased amounts of nucleic acids, and accumulation of collagen and glycosaminoglycans.
EGF with Biobrick standart restriction sites
Oxygen promoter-Vitreoscilla hemoglobin(VHb) promoter in E. coli.
gene of Vitreoscilla hemoglobin(VHb). We synthesized synthetically at GENEART.
The promoter was maximally induced under microaerobic conditions (dissolved oxygen levels of less than 2% air saturation). Transcriptional activity decreased substantially under anaerobic conditions, suggesting the presence of a regulatory mechanism that is maximally induced under hypoxic but not completely anaerobic conditions in E.coli. Primer extension analysis was used to identify the existence of two overlapping promoterswithin a 150-base-pair region upstream of the structural VHb gene. The oxygen-dependent activity of both promoters was qualitatively similar, suggesting the existence of a common mechanism by which available oxygen concentrations influence expression from the two promoters. Oxygen-dependent control mechanisms revealed in some of the above studies include positive regulation by an activator protein. The bacterium Vitreo-scilla sp. is an obligate aerobe from the Beggiatoa family that synthesizes a hemoglobinlike molecule (VHb) in response to growth in oxygen-poor environments. The expression of the VHb gene (vgb) is regulated by oxygen in both its native host, Vitreoscilla, and in E.coli and is maximally induced under microaerophilic conditions (Dikshit and Webster 1988; Dikshit et al. 1992;Joshi and Dikshit 1994). The purposes were to characterize the response of the promoter to changes in oxygen availability in the environment and to obtain initial insights about the mechanism(s) by the promoter is controlled.
The palsmid id pMA (GENEART constantpalsmid with Amphicillin resistance and Biobrick restiction sites,EcoR1,Xba1,Spe1 and Pst1)
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.
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210.
Sequence for pTet inverting regulator driven by the TetR protein.
BBa_R0040 TetR-Regulated Promoter is based on a cI promoter. It has been modified to include two TetR binding sites and the BioBrick standard assembly head and tail restriction sites.
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
tetracycline repressor from transposon Tn10 (+LVA)
Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999)
Coding region for the TetR protein without the Ribosome Binding Site. Modified with an LVA tail for rapid degradation of the protein and faster fall time for the emission. TetR binds to the pTet regulator (BBa_R0040). aTc (anhydrotetracycline) binds to TetR and inhibits its operation.
References (unparsed) here: Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999). Lutz R, Bujard H., Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 1997 Mar 15;25(6):1203-10. PMID: 9092630 References (unparsed) here: Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999). Lutz R, Bujard H., Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 1997 Mar 15;25(6):1203-10. PMID: 9092630 BBa_C0040 TetR Protein is based on the TetR sequence from Elowitz's repressilator. It has been modified to include a rapid degradation LVA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
June Rhee, Connie Tao, Ty Thomson, Louis Waldman.
Conversion of the iGEM parts registry to SBOL2.1