Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: June Rhee, Connie Tao, Ty Thomson, Louis Waldman
Date created: 2003-01-31 12:00:00
Date modified: 2015-08-31 04:07:29
cI(1) fused to tetR promoter
| Types | DnaRegion |
| Roles | promoter Regulatory |
| Sequences | BBa_I1010_sequence (Version 1) |
Description
This part has been optimized for anti-sense inhibition. It is inhibited by anti-sense partsNotes
References (unparsed) here:A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000
Coleman, J., et al. Nature. (1985) 315, 601-3.
Coleman, J., et al. Cell (1984) 37, 429-36.
Pestka, S., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 7525-28.
Jain, C. (1995). IS10 Antisense Control in Vivo is Affected by Mutations Throughout the Region of Complementarity Between the Interacting RNAs. J. Mol. Biol. 246:585-594.
Kittle, J.D., Simons, R.W., Lee, J., and Kleckner, N. (1989). Insertion Sequence IS10 Anti-sense Pairing Initiates by an Interaction Between the 5' End of the Target RNA and a Loop in the Anti-sense RNA. J. Mol. Biol. 210:561-572.
Jain, C. (1997). Models for Pairing of IS10 Encoded Antisense RNAs in vivo. J. theor. Biol. 186: 431-439.
E. coli codon usage table at http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html.
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210
Mizuno, T., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 1966-1970.
References (unparsed) here:
A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000
Coleman, J., et al. Nature. (1985) 315, 601-3.
Coleman, J., et al. Cell (1984) 37, 429-36.
Pestka, S., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 7525-28.
Jain, C. (1995). IS10 Antisense Control in Vivo is Affected by Mutations Throughout the Region of Complementarity Between the Interacting RNAs. J. Mol. Biol. 246:585-594.
Kittle, J.D., Simons, R.W., Lee, J., and Kleckner, N. (1989). Insertion Sequence IS10 Anti-sense Pairing Initiates by an Interaction Between the 5' End of the Target RNA and a Loop in the Anti-sense RNA. J. Mol. Biol. 210:561-572.
Jain, C. (1997). Models for Pairing of IS10 Encoded Antisense RNAs in vivo. J. theor. Biol. 186: 431-439.
E. coli codon usage table at http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html.
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210
Mizuno, T., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 1966-1970.
This protein is built from several parts:
- tetR promoter
- anti-sense binding region, as optimized from references above.
- RBS from references above.
- cI
Anti-senseThe success of this system clearly rests on the ability to effectively and specifically target mRNA transcripts for degradation using anti-sense RNA. While many papers, articles, and books have been written on the subject, there are no consensus anti-sense building strategies presented. We thus chose to implement three different types of antisense inhibition: KISS, micRNA, and IS10. In the description that follows, the following nomenclature will be used:
target- the mRNA transcript that we wish to inhibit.
anti-sense- the anti-sense molecule which will bind and inhibit target.
KISS (Keep it SImple, Silly)
The simplest of the three methods, this type relies on a single-stranded linear 103 bp anti-sense that is specific to the target of interest. In addition, the first 76 base pairs of the cI region of BBa_I1010 have been codon-modified to give a different sequence that codes for the same cI protein (See BBa_I1030 and I1040).
BBa_I1011 contains the reverse complement of the RBS, start codon, and 76 bp region for BBa_I1010. Thus, if both BBa_I1010 and BBa_I1011 are transcribed, the transcripts will bind to each other and BBa_I1010 will not be translated.
Note that BBa_I1010 already contains a regulatory region, RBS, and coding region (a terminator must be added), while BBa_I1011 does not - thus, when using this component, the appropriate regulatory region, RBS, and terminator must be added to this part.
micRNA
This anti-sense mechanism relies on two stem loops flanking an anti-sense sequence that is specific for the target. The function of the stem loops is to maintain the anti-sense region in a quasi-linear state. BBa_I1012 is built in this manner, with a linear region that will bind over the RBS, start codon, and 76 bp of BBa_I1010.
IS10
This method is modeled after the mechanism by which IS10 inhibits production of IS10 transposase. The anti-sense strand is transcribed from the complementary strand of the target (see below), resulting in an anti-sense strand that is 115 bp long, of which 35 bp are complementary to the target. In the absense of a target, these 35 bp form a weak stem loop with the rest of the anti-sense molecule (see below). The key element of the system is the loop at the tip of this stem loop (C-G-G-C-U-U...), which is held in a linear state by the rest of the loop. Upon exposure to the target, the linear loop is able to bind to the 5' end of the target (G-C-C-G-T-T...), and initiate an energetically-favorable zipping/twisting-together of the target and the 5' end of the stem loop (see below). In other words, one side of the weakly stable anti-sense stem loop binds 35 bp of the target, to form a more stable duplex.
I1010 and I1013
Biobricks part BBa_I1013 codes for the exact anti-sense stem loop used in IS10, with two base changes. The 5'-most residues from IS10 anti-sense transcript ( U-C), which do not form part of the stem loop, were changed to G-A. These two bases are reverse-complementary to the first two base pairs of the wildtype cI coding region of BBa_I1010, and thus can bind this region. The rest of the stem loop is wild-type.
The BBa_1010 transcript is targeted by BBa_I1013. The first 35 bases at the 5' end of BBa_I1010 are identical to the first 35 bases at the 5' end of the wild type target, with two differences. Note that three bases T-G-C (which code for cysteine) have been inserted at the 5' end of the cI coding region immediately after the start codon. This allows us to use a wild-type binding pattern at the base of the stem. Since this cysteine is added to the N-terminus of cI, it is not expected to alter the repression ability of cI.
Incompatible with systems containing cI (wild type).
Compatible with systems containing
Source
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000
| Sequence Annotation | Location | Component / Role(s) |
| cI start rbs Modified cI cds TetR 1 -35 TetR 2 -10 start tetR SsrA 2 Complementary to Antisense mRNA added codon (Cys) BBa_I1010 | 82,834 82,84 72,77 85,157 1,19 20,25 26,44 43,48 55,55 1,54 796,834 829,834 55,157 85,87 1,834 | feature/cds CDS start_codon feature/start feature/rbs ribosome_entry_site CDS feature/cds non_covalent_binding_site feature/binding feature/promoter promoter feature/binding non_covalent_binding_site feature/promoter promoter start_codon feature/start promoter feature/promoter feature/tag tag stop_codon feature/stop sequence_feature feature/misc feature/mutation sequence_alteration feature/BioBrick engineered_region |