Our new parts

• J23101x series with modified RBS
• AiiA Expression cassette driven by aTc
• LuxI expression cassette driven by aTc

Existing parts

• RBSx
• 3OC6-HSL biosensor (based on pLux) with different RBSs
• pTet promoter
• AiiA enzime
• LuxI enzyme
• pTet cloning vector

New parts

J23101x series

1. BBa_K516130 (wiki name: J101-E5 ) J101-RBS30-RFP-TT
2. BBa_K516131 (wiki name: J101-31 ) J101-RBS31-mRFP-TT
3. BBa_K516132 (wiki name: J101-E7 ) J101-RBS32-mRFP-TT

BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in 'data analysis' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene.
The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter.
It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P.
This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts. Salis et al. [Nat Biotec, 2009] stated that 'identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels', 'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another'. Attenzione! Non dicono che non è modulare col promotore, ma solo col gene! For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS. In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below. NB: for the RPU computation, the J23101-RBS34-mRFP-tt construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency. If the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.

Existing parts

RBS

The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of mRFP produced. For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. The evaluation of RBS efficiency can be performed in a very intuitive fashion:
-select the RBSs you want to study,
-assemble them in a Promoter - XX - Coding sequence circuit,
-measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS.

This simple measurement system allows the quantification of RBS efficiency depending on the whole measurement system (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in a complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments.
To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.
In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, pTet, pLux). Measuring the system output and evaluating the RBS efficiency. The results are summarized in the table below:

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On the other end, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (pTet-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below: