Revision as of 18:18, 19 October 2019

Trigger 32B

This composite part consists of T7 Promoter (BBa_J64997) and T7 Terminator (BBa_K731721), the Ribosomal Binding Site (AGAGGAGA) and the 32B Trigger (Pardee et al., 2016). Toehold switch systems are composed of two RNA strands referred to as the switch and trigger. The switch RNA contains the coding sequence of the regulated beta lactamase gene. Upstream of this coding sequence is a hairpin-based processing module containing both a strong RBS and a start codon that is followed by a common 21 nt linker sequence coding for low-molecular-weight amino acids added to the N terminus of the gene of interest. A single-stranded toehold sequence at the 5’ end of the hairpin module provides the initial binding site for the trigger RNA strand. This trigger molecule contains an extended single stranded region that completes a branch migration process with the hairpin to expose the RBS and start codon, thereby initiating translation of the downstream gene

Usage and Biology

Documentation

In order to test the sensitivity of our regulatory system we performed experiments by adding different concentrations of trigger and reducing the time of the in vitro protein synthesis reaction. The in vitro transcription/ translation reactions were done using the PURExpress® In Vitro Protein Synthesis kit. A reaction without a trigger sequence was included, as a negative control and a leakage measure. Furthermore, in order to reduce the cost of the reaction, we lowered the reaction volume from 25 to 7 μL.

Firstly, we tested the functionality of our regulatory system by adding following concentrations of trigger:

• 0.3nM (Fig 2.)

• 3nM (Fig 2.)

• 7nM (Fig 1., Fig 2.)

• 75nM (Fig 1.)

Figure 1. Enzymatic assay of beta-lactamase with nitrocefin as its substrate, when expressed from a non-regulated and a toehold-regulated construct in a cell free system. Error bars correspond to standard deviation of n=2 replicates. Blank was subtracted. After the 3-hour incubation in the cell-free system, the chromogenic substrate of β-lactamase, nitrocefin, was added and an additional 45-minute enzymatic assay was performed in the plate reader, at 37oC.

Figure 2. Enzymatic assay of beta-lactamase with nitrocefin as its substrate, when expressed from a non-regulated and a toehold-regulated construct in a cell free system. Error bars correspond to standard deviation of n=2 replicates. Blank was subtracted. Incubation time = 3 hours. After the 3-hour incubation in the cell-free system, the chromogenic substrate of β-lactamase, nitrocefin, was added and an additional 15-minute enzymatic assay was performed in the plate reader, at 37oC

Since PURExpress’ incubation time is the most time-consuming part of our project, we sought to lower the incubation time and assess the performance of the cell-free system and its sensitivity. We performed a 1-hour in vitro protein synthesis reaction, with all the other conditions remaining the same. Again, a gradient of trigger concentrations was included to measure the system’s sensitivity.

The trigger concentrations that were tested are listed below:

• 3nM

• 7nM

• 16nM

• 50nM

• 75nM

After the 1-hour incubation in the cell-free system, we added a chromogenic substrate of β-lactamase, nitrocefin, and performed an additional 15-minute enzymatic assay in the plate reader, at 37oC.

Figure 3. Enzymatic assay of beta-lactamase with nitrocefin as its substrate, when expressed from a non-regulated and a toehold-regulated construct in a cell free system. Blank was subtracted. Incubation time = 1 hour

Sequence and Features