Part:BBa_K325210:Experience
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Applications of BBa_K325210
Measurement by ZJU-China 2022
Introduction
Firefly luciferases (Luc) can catalyze the oxidation of firefly luciferin with molecular oxygen to emit light and are currently being applied as reporter genes for bioimaging and biosensors.[1] The initial reaction catalyzed by firefly luciferase (Luc) is the formation of luciferase-bound luciferyl adenylate (Luc:LH2-AMP) in the presence of Mg2+ and ATP by the release of inorganic pyrophosphate (PPi). The carboxyl group of D-luciferin (LH2) is adenylated. The second step involves the oxygenation of LH2-AMP with molecular oxygen (O2) to produce the excited state of oxyluciferin (Oxyluciferin*), adenosine monophosphate (AMP) and carbon dioxide (CO2). The light emission is produced from the relaxation of excited state oxyluciferin to the corresponding ground state.
Luciferin-regenerating enzyme (LRE) plays an important role in the recycling of oxyluciferin into luciferin, improving the luminescent signal of firefly luciferase, which the following two-step reaction can explain:
(1) Transformation of oxyluciferin to 2-cyano-6-hydroxybenzothiazole
(2) Condensation of 2-cyano-6-hydroxybenzothiazole with D-cysteine to yield luciferin.
So LRE contributes to a reaction recycling oxyluciferin to D-luciferin in the presence of D-cysteine. LRE can be considered as a key enzyme to be applied in luciferase assays.[2]
Here, Luciferase and LRE are expressed, purified and characterized in E.coli. Finally, the kinetics and bioluminescence properties of luciferase are measured.
Materials and Methods
Plasmids and DNA cloning
Using plasmid containing part BBa_K325210 from distribution kits as the template, we separately cloned the coding sequence of luciferase, LRE, LRE and luciferase into pet28a(+) vector. Thus we have three different kinds of constructed plasmid for downstream analyses, called pet28a-luciferase, pet28a-LRE, and pet28a-BBa_K325210. (Fig 1)
Expression and purification of luciferase and LRE
For luciferase and LRE expression, separately transform E.coli BL21(DE3) using pet28a-luciferase, pet28a-LRE and pet28a-BBa_K320214. The transformed E.coli were incubated in 200mL LB medium at 37℃up to OD600=0.6 and then induced at 20℃ with 1mM IPTG for 10h. Cells were harvested by centrifugation at 8000g for 5min at 4℃ and subjected to SDS-PAGE to analyze the whole proteins.
As 6X His tag was added to the N-terminus of exogenous fragments on the vector, the luciferase and LRE were separately purified by agarose-nickel column affinity chromatography. We used His-tag Protein Purification Kit produced by Beyotime (P2226). BL21 with pet28a-luciferase is used to extract purified luciferase, while BL21 with pet28a-LRE is used to extract purified LRE.
(1) The harvested bacteria previously mentioned were suspended in non-denaturing lysate at a ratio of 5ml per gram of bacteria, and the appropriate amount of protease inhibitor mixture was added to the lysate.
(2) Cells were lysed by sonication and centrifuged at 14000g for 15min at 4℃. The supernatant is the crude enzyme solution.
(3) The N-terminal histidine-tagged luciferase and LRE were further purified by agarose-Nickel affinity chromatography. With the mixture in column tube, we collected the flow-through fluid, washed the column 5 times, and eluted the target protein 5 times, collecting each eluate to different centrifuge tubes to obtain purified luciferase or LRE samples[4].
(4) After purification, the flow-through fluid, washing solution and eluate were tested by 10% SDS-PAGE. The concentration of protein was analyzed using the Bradford assay.
Hardware
To test the luciferase bioluminescence intensities, we constructed a hardware, LviSense, which can measure multiple samples with multiple channels at the same time. It can sensitively detect the activity of luciferase and has excellent heat preservation property, which maintains the enzymatic reactions at a specific temperature or under the temperature cycle (Fig 2). Throughout our laboratory work, we use LiveSense to measure luciferase and LRE activity.
For more details, see our Hardware page https://2022.igem.wiki/zju-china/hardware.
Measurement of enzyme activity of luciferase and LRE
To verify that D-luciferin added to the medium can enter the cell through the cell membrane and be catalyzed by the luciferase expressed by E. coli to produce bioluminescence, D-luciferin was added to the cultures of BL21 with vector pet28a-luciferase or pet28a-BBa_K325210 separately, to the final concentration of 0.25 mM. After 10 minutes of incubation, we measured the bioluminescence using LiveSense.
To test the function of LRE catalyzing the regeneration of luciferin, 5μL purified LRE and 5μL purified luciferase were added in triplicate to the reaction mixture containing 0.15mM D-luciferin, 2mM ATP, 10 mM MgSO4 and 5mM D-cysteine in 25mM Tris-HCl (pH=8.0). The total volume of the reaction system is 200μL. For the control group, 5μL LRE was replaced with 5μL 25mM Tris-HCl (pH=8.0)[6][7]. To verify the effect of LRE on luminous intensity and duration, the luminescence signal was measured in triplicate at 25 °C by LiveSense every 10 minutes.
Kinetics measurements and KM determination of luciferase
<p>To quantitatively characterize the ability of luciferase to catalyze bioluminescence, we performed kinetic measurements on luciferase. The enzymic reaction of luciferase has two substrates, luciferin and ATP, so we change concentrations of the two substrates separately in the system, measure luminescence intensity and calculate the Km value. The Km assays for luciferin were performed by mixing 5μL of 40mM ATP/80mM MgSO4 in a solution containing 5μL purified luciferase, 85μL of 0.1M Tris-HCl (pH=8.0) and luciferin at final concentrations between 0.006 and 0.5mM. The Km assays for ATP were performed by mixing 5μL of 80mM MgSO4, in a solution containing 5μL purified luciferase, 85μL of 0.5mM luciferin in 0.1M Tris-HCl (pH=8.0) and ATP at final concentrations in the range of 0.02 and 2.0 mM. All the reagents were added to 96-well plates, incubate for 5 min and measure luminous intensity using "LiveSense". Both assays were performed in triplicate. The Km values were calculated according to the Michaelis-Menten equation.
Results
Expression and purification of luciferase and LRE
The recombinant firefly luciferase was expressed in E.coli and purified by nickel-agarose chromatography. As expected, after IPTG induction, on SDS page the lysate of BL21 with vector pet28a-luciferase shows a specific band of 64.2kDa, which is the band of luciferase. Similarly, the lysate of BL21 with vector pet28a-LRE shows a specific band of 38kDa, which is the band of LRE. Then lysate of BL21 with vector pet28a-BBa_K320214 shows two specific bands of 38kDa and 64.2kDa, which are bands of LRE and luciferase. Both specific bands do not exist in the control group (BL21 without IPTG induction) (Fig 3). The above results demonstrate the successful expression of luciferase and LRE in our engineering bacteria.
Fig 3 expression of luciferase and LRE. A. SDS-PAGE analysis of BL21 with vector pet28a-luciferase. 1: Cell lysate without IPTG induction; 2: Cell lysate-insoluble fraction without IPTG induction; 3:Cell lysate with IPTG induction; 4: Cell lysate-insoluble fraction with IPTG induction. B. SDS-PAGE analysis of BL21 with vector pet28a-BBa_K320214.1: Cell lysate without IPTG induction; 2: Cell lysate-insoluble fraction without IPTG induction; 3:Cell lysate with IPTG induction; 4: Cell lysate-insoluble fraction with IPTG induction.
During the purification of luciferase and LRE, SDS-page analysis was performed on the flowing fluid, washing fluid and eluent. The eluate of luciferase had an obvious band at 64.2kDa, while the eluate of LRE had an obvious band at 38kDa.(Fig 4) These results indicate a good purification effect of luciferase and LRE.
Fig 4 purification of luciferase and LRE. A.SDS-PAGE analysis of purification process of luciferase. B.SDS-PAGE analysis of purification process of LRE.
For both luciferase and LRE, we choose eluent3 as the purified enzyme solution used in the following experiments.
Measurement of enzyme activity of luciferase and LRE
After IPTG induction with luciferin in the culture medium, BL21 cultures with vector pet28a-luciferase and pet28a-BBa_K320214 can both show intense luminescence, while the control without IPTG induction didn't(Fig 5). This pre-experiment verifies that luciferin can enter cells through the cell membrane. Meanwhile, luciferase expressed by BL21 with vector pet28a-luciferase or pet28a-BBa_K325210 can successfully catalyze bioluminescence using luciferin as substrate.
Fig 5 engineered E.coli showing bioluminescence with luciferin in medium. NT: BL21 transformed with vector pet28a-luciferase without IPTG induction; pet28a-luciferase: BL21 transformed with vector pet28a-luciferase under IPTG induction; pet28a-BBa_K320214: BL21 transformed with vector pet28a-BBa_K320214 under IPTG induction.
We examined the luminous intensity at different wavelengths using a fluorescence microplate reader and found that the maximum luminous intensity catalyzed by luciferase appeared at a wavelength of about 600nm(Fig 6).
In the control experiment, the luminescence system with LRE shows lower initial but higher continuous signal than that of the control with luciferase only. The system with functional LRE enzyme complex still generates detectable signals after 12h. Such results show that LRE can catalyze the regeneration of luciferin, but also delay the time to peak luminous intensity, which may be due to competitive binding of luciferin between luciferase and LRE.[5]
Fig 7 Effect of LRE on the in vitro luminous intensity and duration of luciferase. A.Statistical charts of Luminous intensity-Time.x:time after adding luciferase (min); y:luminous intensity. The blue curve represents reaction system with luciferase and LRE, and the red curve represents reaction system with no LRE. B.Change in luminous intensity over incubation time. The upper wells contain reaction systems with luciferase and LRE, and the lower wells contain reaction systems with no LRE.
Kinetics measurements and KM determination of luciferase
In the in vitro luminescence experiment, we found that the KM values for luciferin and for ATP are 0.082mM (Fig 8) and 0.095mM (Fig 9), respectively. However, in most applications, luciferase is used as an in vivo luminescence. In in vivo luminescence experiments, the recommended concentration of D-luciferin is usually 0.25 mM, probably due to the low membrane permeability of D-luciferin. Besides, because sufficient amounts (1~10 mM) of ATP are present in living cells, there is no need to supplement the medium with extra ATP.
Fig 8 Measuring Km values for luciferin. A. Luminous intensity at different concentrations of luciferin. B.Michaelis-Menten equation of luciferase for luciferin, x:1/S (mM-1</p>)(S: concentrations of luciferin), y:1/V (V: relative luminous intensity) C. Luminous intensity - substrate concentration statistical chart, x: S (mM) (concentrations of luciferin), y: V( relative luminous intensity)</p>
Fig 9 Measuring Km values for ATP. AMichaelis-Menten equation of luciferase for ATP, x:1/S (mM-1</p>)(S: concentrations of ATP), y:1/V (V: relative luminous intensity) B. Luminous intensity - substrate concentration statistical chart, x: S (mM) (concentrations of ATP), y: V( relative luminous intensity)
1. Part BBa_K320214, which is the coding sequence of luciferase and LRE, can be expressed properly in E.coli BL21(DE3). The molecular mass of luciferase with tag is about 64.2kDa, while LRE with tag is about 38kDa.
[1] Inouye, S. (2010). Firefly luciferase: an adenylate-forming enzyme for multicatalytic functions. Cellular and molecular life sciences, 67(3), 387-404.
UNIQ9bb4b7252b8ad477-partinfo-00000000-QINU
UNIQ9bb4b7252b8ad477-partinfo-00000001-QINU
Conclusions and Discussion
2. Luciferase can catalyze bioluminescence using luciferin and ATP as substrates, with maximum luminous intensity appearing at a wavelength of about 600nm. The KM values for luciferin and for ATP are 0.082mM and 0.095mM.
3. The luciferin-regenerating enzyme(LRE) can catalyze the regeneration of luciferin from oxyluciferin, thus extending the glow time. Therefore, LRE is supposed to provide a more stable luminescent signal together with the catalyzing of luciferase.
4. We have created a reliable hardware "LiveSense" as a high-throughput instrument to measure the bioluminescence catalyzed by luciferase.
Reference
[2] Gomi, K., & Kajiyama, N. (2001). Oxyluciferin, a luminescence product of firefly luciferase, is enzymatically regenerated into luciferin. Journal of Biological Chemistry, 276(39), 36508-36513.
[3] Moreira, A. C., Amaral, D. T., Gabriel, G. V. M., & Viviani, V. R. (2022). Cloning and molecular properties of a novel luciferase from the Brazilian Bicellonycha lividipennis (Lampyridae: Photurinae) firefly: comparison with other firefly luciferases. Photochemical & Photobiological Sciences, 1-13.
[4] Sun, X., Tang, X., Hu, R., Luo, M., Hill, P., & Fang, B. (2019). Biosynthetic bifunctional enzyme complex with high-efficiency luciferin-recycling to enhance the bioluminescence imaging. International journal of biological macromolecules, 130, 705-714.
[5] Hemmati, R., Sajedi, R. H., Bakhtiari, N., & Hosseinkhani, S. (2014). Directed improvement of luciferin regenerating enzyme binding properties: implication of some conserved residues in luciferin‐binding domain. Photochemistry and Photobiology, 90(6), 1293-1298.
[6] Sun, X., Tang, X., Wang, Q., Chen, P., Hill, P., & Fang, B. (2018). Fusion expression of bifunctional enzyme complex for luciferin-recycling to enhance the luminescence imaging. Journal of Photochemistry and Photobiology B: Biology, 185, 66-72.
[7] Emamzadeh, R., Hosseinkhani, S., Hemati, R., & Sadeghizadeh, M. (2010). RACE-based amplification of cDNA and expression of a luciferin-regenerating enzyme (LRE): An attempt towards persistent bioluminescent signal. Enzyme and Microbial Technology, 47(4), 159-165.
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