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Biobricks

 

Design Goal Methods References

Applications Link

Background
For more information about Synthetic Biology visit the Synthetic Biology web pages at MIT.

For more information about Biobricks visit the Biobricks page.

For more information about the repressilator system see this article. 

Design Goal

To improve upon Elowitz's repressilator. We chose to implement this improvement by decreasing the period using an anti-sense RNA interference step. The Interfering Repressilator is designed to exhibit oscillatory behavior in the form of periodic expression of Yellow Fluorescent Protein (YFP).


Description of System

Simplified Design

Biological Representation

Parts List

BB Part Description
Basic Parts
BBa_B0002 Base Plasmid for Structured Assembly
BBa_B0012 Terminator (Double Stop Codon)
BBa_B0030 Ribosome Binding Site (strong)
Coding Sequences for Proteins
BBa_C0012 LacI Protein
BBa_C0040 TetR Protein
BBa_E0022 Cyan Fluorescent Protein w/LVA Tail w/o RBS
BBa_E0032 Yellow Fluorescent Protein w/LVA Tail w/o RBS
Custom-Designed Parts
BBa_I1010 cI Fused to TetR Promoter
BBa_I1011 Anti-sense RNA (KISS)
BBa_I1012 Anti-sense RNA (micRNA)
BBa_I1013 Anti-sense RNA (IS10)
Regulatory Parts
BBa_R0011 LacI Promoter
BBa_R0040 TetR Promoter
BBa_R0050 HK022 cI Promoter
BBa_R0051 Lambda cI Promoter

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)

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.

Methods

Simulations

We simulated our model using Matlab. Differential equations were developed to model the change in concentration of 5 key species: cI mRNA, lacI mRNA, anti-sense mRNA (asRNA), cI protein, and lacI protein. The model uses the parameters from Elowitz (2000). The only other parameter that was required was the rate constant for mRNA/asRNA binding. This rate constant was about 1E6 M-1s-1 (Jain, 1997), which translates to 2E4 molecule-1s-1.

Summary

It doesn't oscillate. That's it. Unfortunately, it was determined that our design is very sensitive to the rate constant for mRNA/asRNA binding. If this constant is too low (ie below 0.02 molecule-1s-1) then the system fails to oscillate for the values of the other constants as given by Elowitz. With the constant at its expected value (two orders of magnitude below this critical value) the system fails to oscillate for all values of the other constants within several orders of magnitude. It appears that this rate constant is the critical piece in our design, and we were not able to come up with strategies to get around this problem.

Graphs


Source Codes

Main Program Code

Function Code

Important Parameters (and Links to Articles from which gathered)

As described in the summary section, the critical parameter is the rate constant for mRNA/asRNA binding. It appears to limit the performance of our design, for which no other reasonable range of values of the other constants could compensate.

Challenges and Debug Plan

(Why it may or may not work) BlahBlahBlahBlahBlahBlah

Debug Plan to make it work BlahBlahBlahBlahBlahBlah

Future Research

Comments for future projects groups

References

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.

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

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.

Jain, C. (1997). Models for Pairing of IS10 Encoded Antisense RNAs in vivo. J. theor. Biol. 186: 431-439.

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.

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

Mizuno, T., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 1966-1970.

Pestka, S., et al. Proc. Natl. Acad. Sci. USA (1984) 81, 7525-28.

Group Members
June Rhee
Connie Tao
Ty Thomson
Louis Waldman