**2. Materials and methods**

### **2.1 Dictyostelium discoideum**

Under the National BioResource Project (NBRP), the National Institute of Advanced Industrial Science and Technology (AIST) in Japan provided the *Dictyostelium discoideum* strain Ax2 (NBRP ID: S00001). The Ax2 cells were cultured in SM/5 medium on a 1.4% agar plate at 21°C with *Klebsiella aerogenes* bacterial cells (provided by Prof. H. Kuwayama, Tsukuba University, Japan) as feed.

#### **2.2 Firefly luciferase gene**

Green- and red-emitting luciferases were used for the dual-color bioluminescence promoter assay. The green-emitting luciferase gene *Luci sp1* was cloned from *Luciola* sp. collected in the Belum forest, State Park, Malaysia. *Luci sp1* was modified and optimized for mammalian cell expression. Variant 1 of *Luci sp1* [17] (DNA Data Bank of Japan [DDBJ] accession no. LC632706) was used for the green vector construction. The red-emitting luciferase gene *Psa* (wild-type) was cloned from *Pristolycus sagulatus* collected in Tokyo, Japan. *Psa* (Wildtype) was modified and optimized for mammalian cell expression as *Psa* [18] (DDBJ accession no. LC495933). This luciferase gene was used for the red vector construction and was also deposited in the RIKEN BioResource Research Center (BRC), Tsukuba, Japan (BRC catalog no. RDB14361).

#### **2.3 Construction of** *adenylyl cyclase A* **reporter vector**

The *Dictyostelium* extrachromosomal expression vectors pDM304 (NBRP ID: G90008) and pDM358 (NBRP ID: G90009) were provided by Tsukuba

*Imaging Promoter Assay of Adenylyl Cyclase A Gene in* Dictyostelium discoideum*… DOI: http://dx.doi.org/10.5772/intechopen.101485*

University under the NBRP and were used in the construction of two *acaA* reporter vectors involving promoters 1 and 3. The actin 15 promoter (*Xho*I/*Bgl*II restriction sites) of the pDM358 and pDM304 vectors was replaced with promoters 1 and 3 of *acaA*, respectively. The promoter regions were amplified using PCR from *Dictyostelium discoideum* Ax2 genomic DNA using the primer sets for promoter 1 (5′-GCctcgagCTTGATGAGTGGCCCAAAACC-3′ and 5′-GCagatctATTTTTAAAGATCCAAGAATTCG-3′) and promoter 3 (5′-GCctcgagACCTCACTTCATAAATATATCTTTG-3′ and 5′-GCagatct TTTTTAATAATTTTTTAATATTATTAC-3′) [10]. Variant 1 of *Luci sp1* (green) and *Psa* (red) luciferase genes, including the *Dictyostelium* Kozak sequence (AAAA) before the start codon, was inserted into downstream of the promoter in the pDM358 and pDM304 vectors, respectively, *via* the *Spe*I/*Hind*III restriction sites. These vectors contained the actin 8 terminator.

### **2.4 Transformation and fruiting body formation**

The Ax2 cells were co-transfected with the two constructed vectors by electroporation using a MicroPulser (Bio-Rad, California, USA) according to the protocol for *Dictyostelium*. The transformant cells were selected using hygromycin (50 mg/mL) and neomycin (10 mg/mL) in HL5 medium.

Millicell Cell Culture Insert PICM 0RG50 (Merck, Darmstadt, Germany) was placed onto a 35-mm glass bottom dish, and 1.2 mL of D buffer (saline solution for *Dictyostelium*) containing 3 mM D-luciferin potassium salt (Promega, Wisconsin, USA) was added to the basolateral side of the glass bottom dish. The transformant cells were seeded onto the inside of the cell culture insert above the membrane and cultured at room temperature (21°C), facilitating the development of the mound, slug, and fruiting body.

#### **2.5 Bioluminescence microscopy**

The bioluminescence images of the cells were captured using an LV200 bioluminescence microscope (Olympus, Tokyo, Japan) [19, 20] equipped with a UCPLFLN 20XPH objective lens (Olympus) and an ImagEM C9100-13 EM-CCD camera (Hamamatsu Photonics, Shizuoka, Japan). The activities of *acaA* promoters 1 and 3 were visualized using BA495-540GFP (Olympus) and 610ALP (Omega, New Jersey, USA) emission filters, respectively, at an exposure time of 30 s for 24 h at 90s intervals. In addition, bright-field images were captured at an exposure time of 200 ms using the same capture sequence as that of the bioluminescence images. The time course of luminescence intensity was analyzed using TiLIA, a time-lapse image analysis software [21].

#### **2.6 Fluorescence microscopy**

The autofluorescence images of the cells were captured using an IX83 inverted microscope (Olympus) equipped with a U-HGLGPS excitation light source (output level 100 with ND25 filter), a U-FGFP mirror unit, and a DP74 color CCD camera. A UCPFLN 10XPH objective lens was used for mound and slug observations, and a UCPFLN 4xPH objective lens was used for fruiting body observation. Exposure time was 500 ms for all experiments.

The irradiation power of the excitation light was measured at 480 nm using a PM100D optical power meter (Thorlabs, New Jersey, USA) with an S170C sensor probe for microscopy.
