Difference between revisions of "Part:BBa I1010:Design"

Latest revision as of 14:24, 21 July 2006

cI(1) fused to tetR promoter

Design Notes

E. coli codon usage table at http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html.

This protein is built from several parts:

- tetR promoter BBa_R0040

- anti-sense binding region, as optimized from references above.

- RBS from references above.

Anti-sense

The 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) <img src="http://biobricks.ai.mit.edu/IAP_Projects/YoungPower/as_KISS.gif">

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 <img src="http://biobricks.ai.mit.edu/IAP_Projects/YoungPower/as_micRNA.gif">

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 <img src="http://biobricks.ai.mit.edu/IAP_Projects/YoungPower/as_IS10.gif">

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 BBa_I1020, BBa_I1021, BBa_I1022, BBa_I1023.

Source

Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210

<a href="http://www.nature.com/cgi-taf/DynaPage.taf?file=/nature/journal/v403/n6767/abs/403335a0_fs.html&dynoptions=doi1043774228">A synthetic oscillatory network of transcriptional regulators</a> , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000

References

References (unparsed) here:

• <ahref="http://www.nature.com/cgi-taf/DynaPage.taf?file=/nature/journal/v403/n6767/abs/403335a0_fs.html&dynoptions=doi1043774228">A synthetic oscillatory network of transcriptional regulators</a> , 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.
• Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210
• Mizuno, T., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 1966-1970.