Part:BBa_K2973007

32B Toehold Switch_β-lactamase_no signal peptide

This composite part consists of T7 Promoter (BBa_J64997) and T7 Terminator (BBa_K731721), the Ribosomal Binding Site (AGAGGAGA), the 32B Toehold Switch (Pardee et al., 2016) and the CDS of the β-lactamase without the signal peptide. 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 β-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 β-lactamase. 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 amid 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 yellow color, but after its hydrolysis by β-lactamase , the color of the solution turns red, allowing in that way the detection of the enzyme.

Improvement

This part is an improved version of BBa_K1189007.

Aim

Our project design, utilizes the ability of the β-lactamase enzyme to hydrolyze the chromogenic substrate, nitrocefin, resulting in a color change from yellow to red. Based on this ability of β-lactamase, we decided to improve an already existing Biobrick (BBa_K1189007) which corresponds to the β-lactamase gene regulated by an inducible lacI promoter. This Biobrick part is only functional in vivo, and cannot be expressed with any of the usual in vitro transcription translation kits, because of the lack of an appropriate promoter (recognized by either T3, T7, or SP6 polymerase). Aiming to fit this part into our cell-free system and make it functional in vitro, we firstly replaced the lacI, inducible by IPTG, promoter with a T7 constitutive promoter (BBa_J64997). In order to regulate its expression, just as the lacI promoter does, we incorporated a Toehold switch sequence upstream of the CDS of β-lactamase. To be able to prove our improvement upon this part, we used the chromogenic substrate nitrocefin, which changes color from yellow (380nm) to red (490nm) when hydrolyzed by the β-lactamase enzyme. We then gathered quantitative results for the hydrolysis of our substrate, Nitrocefin, by both parts, though frequent plate reader assays after both in vivo and in vitro expression of the above mentioned constructs.

Constructs' creation

For our experiments, we designed the composite part BBa_K2973007 that consists of a T7 promoter, Pardee’s Toehold Switch 32B [1], a β-lactamase gene and a T7 terminator. Furthermore, we designed the composite part BBa_K2973023 that consists of a T7 promoter, Pardee’s Trigger 32B, and a T7 terminator. These parts, including the prefix & suffix sequences, were ordered from IDT and cloned into their respective plasmids (pSB1K3 vector for the 32B Trigger and pSB1C3 for the lacI β-lactamase and the 32B Toehold β-lactamase), with restriction digestion & ligation.

In vivo protein expression assay

Our first goals for our improvement experiments, was to demonstrate that our improved part can be functional in vivo, after activation from our trigger sequence, and also prove that it is able to reach the expression levels of the original part (BBa_K1189007).

Method

In order to produce measurable and reproducible data, we used 2 biological and 2 technical replicates for each construct. Aiming to test the binding efficiency between the Toehold switch regulating the β-lactamase enzyme and the Trigger32B, we co-transformed the two plasmids that contained these constructs into BL21 (DE3) cells. Moreover, BL21 (DE3) cells were transformed with the plasmid that contains the lacI-regulated β-lactamase construct, in order to compare our improvement hypothesis.

To ensure that the absorbance measured corresponds only to the enzymatic activity of β-lactamase, we included 5 controls in our experiments:

• LB medium only (no cells) and nitrocefin

• Empty BL21 (DE3) cells (no plasmid) and nitrocefin

• BL21 (DE3) cells containing the empty vector and nitrocefin

• BL21 (DE3) cells with Toehold32B- β-lactamase and nitrocefin

• BL21 (DE3) cells with lacI- β-actamase (BBa_K1189007) and nitrocefin

The workflow of our in vivo experiments was performed as described below:

1. We grew the cultures overnight in 5ml LB (~16h) at a shaking incubator, 37℃ / 210rpm

2. The following morning, we measured the OD600 of the overnight cultures

3. We diluted all cultures to OD600 = 0.1 in LB medium

4. We then grew the cells at 37℃ /210 RPM until OD600=0.5 (~2h)

5. We diluted all cells to the same OD600 (e.g. 0.5)

6. We loaded 200ul of culture in a 96-well plate (2 technical replication each) and 40ul of the nitrocefin substrate (0,5 mM), in order to perform the enzymatic assay.

7. We finally measured the absorbance at 490nm (for nitrocefin hydrolysis) and 600nm (for cell growth) in a microplate reader. We shook between measurements.

The absorbance measurements were conducted every 2 min for 75 minutes at 490nm for the hydrolyzed nitrocefin substrate, and at 600nm for the cell growth.

Results

Figure 1. Expression of β-lactamase reporter gene in vivo. Error bars represent the standard deviation for n = 2 biological replications. The substrate (nitrocefin) hydrolysis (490nm) is divided by cell growth (600nm), in order to normalize all values.

Figure 2. β-Lactamase expression levels for t=75 minutes. Error bars represent the standard deviation for n = 2 biological replications.The substrate (nitrocefin) hydrolysis (490nm) is divided by cell growth (600nm), in order to normalize all values.

Figure 3. Change of the cultures’ color from yellow to red due to the hydrolysis of nitrocefin ( in vivo )

In vitro protein expression assay

For the second part of our improvement experiments we wanted to demonstrate that our improved part is not only functional in vivo but also in cell-free systems. Furthermore, we proved that the initial part (LacI-regulated β-lactamase) is not functional in vitro and cannot be expressed with our in vitro translation kit (PURExpress In vitro Protein Synthesis Kit), as it cannot be expressed with any of the usual in vitro transcription translation kits due to the lack of an appropriate promoter (recognized by either T3, T7, or SP6 polymerase).

Method

In order to produce measurable and reproducible data, we used 2 technical replicates for each construct. Τhe constructs that were used during the experiment and their respective quantities are listed below:

• T7 β-lactamase (positive control) 75nM

• Toehold 32B β-Lactamase (negative control) 75nM

• Toehold 32B β-Lactamase 70ng + Trigger 32B 75nM

• Toehold 32B β-Lactamase 70ng + Trigger 32B 7nM

• LacI β-Lactamase (initial part) 75nM

Our in vitro experiments were performed with the PURExpress Ιn Vitro Protein Synthesis Kit provided by New England Biolabs (NEB). We followed the standard protocol for a typical 7ul PURExpress reaction. Each PURExpress reaction was incubated for 3 hours at 37 ℃ for each construct. Finally, we measured the absorbance levels of our samples every 30 seconds for 45minutes total, at 490nm to be able to have quantitive results for the hydrolysis of our substrate (Nitrocefin).

Results

Figure 4. Expression of β-lactamase reporter gene in vitro. Error bars represent the standard deviation for n = 2 technical replications.

Figure 5. Colour-change from yellow to red due to the hydrolysis of nitrocefin (in vitro).

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 7.)

• 3nM (Fig 7.)

• 7nM (Fig 6., Fig 7.)

• 75nM (Fig 6.)

Figure 6. Enzymatic assay of β-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 37℃.

Figure 7. Enzymatic assay of β-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 37℃

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 37℃.

Figure 8. Enzymatic assay of β-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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]