Team:Thessaly/Improve
IMPROVEMENT.
During our project, we decided to improve an already existing BioBrick that encodes for the β-lactamase enzyme, coupled with a His-tag, under the control of the IPTG inducible lacI promoter. By altering its sequence and adding a new regulatory system, a toehold switch, we managed to regulate its function both in vivo and in vitro and generate a new part that can be functional also both in vivo and in vitro. The methods we followed in our experiments and the results of our experiments are presented in this page.
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 abovementioned constructs.
Constructs' engineering
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.
Table 1. The Registry Parts used for the generation of our newly designed BioBricks for our improvement experiments.
In vivo protein expression assay
Initially, our goals for the improvement experiments were to demonstrate that our improved part can be functional in vivo after activation from our trigger sequence, and that it is also 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.
The graph above shows the hydrolysis of our substrate (nitrocefin) absorbing at 490nm divided by cell growth at 600nm, to normalize all values. Every value accrues from the subtraction of the values of our Blank controls (Nitrocefin + water) from the initial absorbance given by the constructs. Since all conditions remain the same, differences in the curves indicate different expression levels of the β-lactamase constructs.
The lacI-β-lactamase construct (yellow line) reaches a plateau phase after approximately 35 minutes due to substrate limitation. After 75 minutes our improved part (green line), Toehold 32B β-lactamase which is activated by the 32B trigger, reaches the same expression levels as the positive control. Finally, the negative controls’ lines (purple, blue, grey) confirm that the measured absorbances derive from β-lactamase’s activity only.
The graph below shows the difference between the absorbance levels of the lacI regulated β-lactamase part and our toehold switch system. At t=75minutes, the absorbance levels of our improved part reach approximately the same levels as the lacI-regulated β-lactamase part. On the other hand, both the toehold 32B β-lactamase (no trigger), shown in green, and the negative control (BL21 DE3 cells with empty plasmid), shown in purple, don’t produce any enzyme and that’s why their respective absorbance levels are significantly low.
Figure 2. β-Lactamase expression levels for t=75minutes. 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 original 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 75nM + trigger 32B 75nM
• Toehold 32B β-Lactamase 75nM+ trigger 32B 7nM
• LacI β-Lactamase (original 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 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 hydrolyzation 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.
The graph above depicts the substrate (nitrocefin) hydrolysis (490nm). The T7 β-lactamase construct (blue line) reaches a plateau phase after approximately 5 minutes due to substrate limitation. The absorbance levels of the two different quantities of the trigger, 75nM, and 7nM, which were used are depicted with a grey line for the 75nM and with a light blue line for the 7nM. The absorbance levels of the 75nM trigger used, are significantly similar to the levels of our positive control (T7 β-Lactamase). In that way, we prove that our toehold switch is functional also in vitro, as a regulatory system and is very efficient at expressing the β – Lactamase enzyme. Moreover, the light blue line (7nM), proves that our toehold switch system can be also activated by lower than 75nM concentrations of our trigger sequence. Finally, the original part (lacI-regulated β-lactamase), shown in green color, isn’t expressed in vitro as expected.
Figure 5. Color-change from yellow to red due to the hydrolysis of nitrocefin (in vitro)
Conclusion
In conclusion, our improved part, BBa_K2973007, consisted by the β-lactamase gene regulated by a toehold switch and a T7 promoter is efficiently expressed and is functional both in vivo and in vitro. Instead, the original part, BBa_K1189007, consisted by the β-lactamase gene under the control of an IPTG inducible lacI promoter, can be expressed only in vivo. In this way, we managed to generate a new part that can be functional both in vitro and in vivo, by changing its promoter and introducing a new regulatory system for gene expression.
References
1. Pardee, Keith, et al. “Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components.” Cell, vol. 165, no. 5, 2016, pp. 1255–1266., doi:10.1016/j.cell.2016.04.059.
