Difference between revisions of "Part:BBa K3202068"
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===Design===
 
===Design===
  
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In our system(Fig. 1), we have transcriptional repressors R1 and R2 expressed through the inducible promoter but translationally inhibited by the MicC sRNA1 and sRNA2 respectively, since RBS2 and RBS3 is directly inhibited by sRNA while they initiates the transcription of the two repressors; the gene of interest is translationally coupled to the repressor gene while three promoters which produces the inhibitory sRNA are targeted by their corresponding repressors. Therefore, repressors and sRNAs are mutually inhibitory.
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In our system(Fig. 1), we have transcriptional repressors R1 and R2 expressed through the inducible promoter but translationally inhibited by the MicC sRNA1 and sRNA2 respectively, since RBS2 and RBS3 is directly inhibited by sRNA while they initiates the transcription of the two repressors; the gene of interest is translationally coupled to the repressor gene while three promoters which produces the inhibitory sRNA are targeted by their corresponding repressors. Therefore, repressors and sRNAs are mutually inhibitory.  
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[[File: T--BHSF ND--Bistable design 1.png |350px|thumb|center|alt=Basic Design of Bistable System|Figure 1]]
  
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[[File:T--BHSF ND--Bistable design 1.png |500px|thumb|none|alt=TU.|Figure 1. Transcriptional unit assembled. Golden Gote scars: standardized overhangs for promoter, RBS, CDS and terminator]]
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 13:59, 20 October 2019


AraC-Pc-pBAD-RBS1-Colicin E2-T500-T1/TE-MicCsRNA-PA1/O4

This composite part is designed to insert the toxin Colicin E2 into the bistable system.

Presenting with BBa_K3202066-BBa_K3202067, this part is to test the function of the toxin within our bistable system.

Background

We are inspired by a lately published article written by Belén Calles, Angel Goñi-Moreno, and Víctor de Lorenzo to design a double-bistable system which achieves zero basal expression of gene of interest in the absence of any inducer while ensuring constant transcriptional capacities and induction rates among promoters.

“The mechanism of a bistable system is elaborated below: a repressor transcriptionally inhibits the promoter which is responsible for the transcription of sRNA, while the sRNA translationally inhibits the repressor. The gene of interest is translationally coupled to the repressor gene. That is to say, the repressor protein R targets the strong promoter which the inhibitory sRNA is produced, so that R and sRNA are mutually inhibitory. Such arrangement helps to minimize the basal activity of the inducible module and thus suppress leaky gene expression levels.”

Design

In our system(Fig. 1), we have transcriptional repressors R1 and R2 expressed through the inducible promoter but translationally inhibited by the MicC sRNA1 and sRNA2 respectively, since RBS2 and RBS3 is directly inhibited by sRNA while they initiates the transcription of the two repressors; the gene of interest is translationally coupled to the repressor gene while three promoters which produces the inhibitory sRNA are targeted by their corresponding repressors. Therefore, repressors and sRNAs are mutually inhibitory.

Basic Design of Bistable System
Figure 1


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961