Revision as of 17:55, 19 October 2019

T7-RBS-β-Lactamase-No Signal peptide

This composite part consists of T7 Promoter(BBa_J64997) and T7 Terminator(BBa_K731721), Ribosomal Binding Site(BBa_B0034), CDS of β-lactamase without the signal peptide. β-lactamase (EC 3.5.2.6) is a small monomeric enzyme(29kDa) that is produced by bacteria and gives them resistance to antibiotics with β-lactam ring because of its ability to hydrolyze the amide bond in the β-lactam ring. This ability can be exploited in order to use β-lactamase as a protein reporter by providing the enzyme with its chromogenic substrate Nitrocefin. Nitrocefin is a chromogenic cephalosporin first reported in 1972 as a novel and straightforward substrate used to detect bacteria resistant to β-lactam antibiotics. Normally, a nitrocefin solution has a yellow color, but after its hydrolysis by β-lactamase, the color of the solution turns red, allowing that way the detection of the enzyme.

Usage and Biology

Documentation

This part was used as a positive control for our in vitro protein synthesis experiments.

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.)

After the 3-hour incubation in the cell-free system, we added a chromogenic substrate of β-lactamase, nitrocefin, and performed an additional 45-minute enzymatic assay in the plate reader, at 37oC.

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.

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.

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. Error bars correspond to standard deviation of n=2 replicates. Blank was subtracted. Incubation time = 1 hour.

Sequence and Features