Part:BBa_K3202068

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.

Figure 1

RBS1

The control of the recombinase in coordination with the upstream repressor gene, which is the target of the sRNA, requires a translational coupler cassette. “An efficient mechanism to reach this goal is based on the ability of translating ribosomes to unfold mRNA secondary structures. In our design, translational coupling is achieved by occluding the RBS of gene of interest by formation of a secondary mRNA structure, containing a His-tag sequence added to the 3 ́end of the repressor gene. The hairpin structure prevents the ribosomal recruitment to the recombinase and therefore its translation. In contrast, when the upstream repressor mRNA is actively translated, the 70s ribosome disrupts inhibitory mRNA secondary structure in the downstream gene translation initiation region thus allowing its expression.”

However, during the experiment we tested that the RBS1 (Fig. 2) sequence provided by the original article fails to function in our circuit, we found RBS2 (Fig. 3) with a different working mechanism. The structure of AUGA in RBS2 acts as both the stop codon (AUG) of the former gene sequence, releasing the former peptide chain; takes a step (gene code) backward, pinpointing a new ribosome binding site, and initiated the translation of next gene sequence with the start codon (UGA). However, finally we still used RBS1 since the crux actually lied in another place.

Figure 2

Figure 3

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