Part:BBa_K325210

Red Firefly Luciferase and LRE
L. Cruciata
(E. coli optimised)

This part is contains the coding sequence for a mutant light-emitting enzyme (luciferase) and luciferin regenerating enzyme (LRE) from the Japanese firefly, Luciola cruciata. It will make bacteria emit light at a reddish wavelength when placed under a promoter, provided the media is supplemented with luciferin.

The luciferin regenerating enzyme will convert oxyluciferin into CHBT. CHBT is converted to more luciferin in the presence of D-cysteine.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2060
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 951
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 900
  Illegal AgeI site found at 2602
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 332
  Illegal SapI.rc site found at 2116
  

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.