Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Claudia Ivonne Hernandez Armenta
Date created: 2010-09-08 11:00:00
Date modified: 2015-05-08 01:12:12
RBS + LovTAP Photosensor + terminator
| Types | DnaRegion |
| Roles | Signalling engineered_region |
| Sequences | BBa_K360121_sequence (Version 1) |
Description
This photoreceptor was assembled by Strickland et al., and consists of a LOV (light-oxygen-voltage) domain of Avena sativa phototropin1 (AsLOV2) that senses blue light, fused to the trpR- DNA binding domain of the transcription factor trp repressor. The resulting protein is named LovTAP: LOV- and tryptophan-activated protein.LOV domains bind a flavin-mononucleotide (FMN) or flavin-adenine-dinucleotide (FAD) cofactor, which are used in a wide variety of metabolic pathways as cofactors in redox reactions and are available in most organisms. The cofactor has a broad absorption spectrum, with a maximum at 450 nm. Besides, the core of the LOV domain is often flanked by amino- or carboxy-terminal helices, termed A???α and Jα, respectively
In the LovTAP construction, AsLOV2 domain via its carboxyl-terminal Jα ???helix was ligated to an amino-terminal truncation of TrpR. The resulting protein has a domain-domain overlap with a shared helix. Thus, photoexcitation would change the conformation of the protein, in turn changing the stability of the shared-helix-domain contacts.
Under the presence of light, absorption of a photon leads to the formation of a covalent adduct between the flavin mononucleotide (FMN) cofactor and a conserved cysteine residue in the AsLOV2 domain, which results in conformational rearrangements in LovTAP. This change impacts the affinity of the shared helix for the two domains: disrupting the contacts between the shared helix and the LOV domain and enabling the association of the shared helix with the TrpR domain, which establishes DNA-binding affinity and LovTAP can then bind DNA as an homodimer, repressing the transcription of the genes downstream of its binding sites.
In the dark, when the shared helix contacts the LOV domain, the TrpR domain's DNA-binding affinity decreases and LovTAP is in an inactive conformation.
Notes
We decided to synthesize a new LovTAP part, that in comparison with the Part:BBa_K191003 that is already at the registry, has the following differences:1. The 2 PstI restriction sites were removed from the coding region of LovTap.
2. We included a punctual mutation to change the ILE427 by a PHE427, as was proposed by the model results of the team iGEM09_EPF-Lausanne. With this mutation LovTAP should react faster and the conformational change should be more stable (the protein stays in the active form for longer, under light induction).
3. The part does not include a promoter. We eliminated the inverting regulator sensitive to LacI and CAP protein(Part:BBa_R0010). According to the report of EPF-Laussane team, the expression levels of LovTAP under the inverting regulator, doesn??t seem to show differences to the induction with IPTG. Thus, we decided to remove it and as we know that the level expression of LovTAP must be low (because at high levels of expresion, there is unspecific regulation under darkness and light states), we plan to test weak promoters to choose the best option and include it in the construction.
4. The RBS was changed from a strong (Part: BBa_B0030) to a medium strength (Part:BBa_B0032), thus expecting lower levels of lovTAP protein.
5. The stop codon tga was changed for two taa.
Source
Chiameric protein assembled by Strickland et al.Reference:
Strickland, D., Moffat, K., & Sosnick, T. (2008). Light-activated DNA binding in a designed allosteric protein. Proceedings of the National Academy of Sciences, 105(31), 10709. National Acad Sciences. Retrieved from http://www.pnas.org/content/105/31/10709.full.
| Sequence Annotation | Location | Component / Role(s) |
| RBS B0032 stop codon LovTAP K360022 Terminator B0010 Terminator B0012 AAA | 1,13 702,706 18,701 725,771 804,830 831,844 | ribosome_entry_site feature/rbs feature/stop stop_codon CDS feature/cds feature/stem_loop stem_loop feature/stem_loop stem_loop feature/polya polyA_site |