dsred2 cdna (egfp cdna for mkate2)  (TaKaRa)

Journal: ACS Central Science

Article Title: Edible Matrix Code with Photogenic Silk Proteins

doi: 10.1021/acscentsci.1c01233

Figure Lengend Snippet: Fabrication of edible matrix codes using silk fibroin genetically hybridized with fluorescent proteins. (a) Schematic representation of transformation vector structures for silkworm transgenesis, p3×P3-DsRed2-pFibH-eCFP for eCFP silk, p3×P3-DsRed2-pFibH-eGFP for eGFP silk, and p3×P3-eGFP-pFibH-mKate2 for mKate2 silk. Fibroin heavy chain promoter domain (pFibH, 1124 base pairs (bp)), N-terminal region 1 (NTR1, 142 bp), Intron (871 bp), N-terminal region 2 (NTR2, 417 bp), C-terminal region (CTR, 179 bp), poly(A) signal region (PolyA, 301 bp), enhanced cyan fluorescent protein (eCFP, 720 bp), enhanced green fluorescent protein (eGFP, 720 bp), monomeric far-red fluorescent protein (mKate2, 699 bp), inverted repeat sequences of piggyBac arms (ITR), 3×P3 promoter (273 bp), and Sv40 polyadenylation signal sequence (Sv40pA, 268 bp). Red fluorescent protein (DsRed2) is used only for a marker gene of eCFP and eGFP, while eGFP is utilized for a marker gene of mKate2. (b) Photographs and fluorescence images of eCFP silk, eGFP silk, and mKate2 silk cocoons, compared with a nontransgenic (wild-type) white silk cocoon. Each silk fibroin solution is regenerated from the corresponding silk cocoons. (c, d) Optical absorption (c) and fluorescence emission (d) spectra of fluorescent silk fibroin films fabricated using the regenerated eCFP silk (cyan), eGFP silk (green), and mKate2 silk (red) fibroin solutions. (e) Photographs and fluorescence images of three different fluorescent silk fibroin films and a white silk fibroin film using an appropriate set of optical excitation and emission. A set of an excitation source (λ ex ) and an emission filter (λ em ) is used as follows: λ ex = 415 nm and λ em = 460 nm, λ ex = 470 nm and λ em = 525 nm, and λ ex = 530 nm and λ em = 630 nm for eCFP silk, eGFP silk, and mKate2 silk, respectively. The thickness of the fluorescent silk fibroin films is 70 μm on average. (f) Scanning electron microscopy images of conical micrograting arrays with 2D periodic hexagonal patterns. The height and bottom diameter of each grating are 1.4 and 2.7 μm with a distance (i.e., period) between adjacent gratings of 2.9 μm. (g) Photographs of light propagation (green laser at λ = 532 nm) through bare (top) and micrograting patterned (bottom) silk fibroin films. (h) Photograph of 7 × 7 matrix codes fabricated using bare (left) and micrograting patterned (right) silk fibroin films, affixed onto the tablet-type medicine (oral solid dosage). The code pattern on the micrograting patterned silk fibroin film is covert and imperceptible due to the strong diffraction of light caused by the micrograting arrays.

Article Snippet: To construct the plasmids, the marker DsRed2 cDNA (eGFP cDNA for mKate2) was amplified by polymerase chain reaction (PCR) using specific primers with Nhe I/ Afl II sites from pDsRed2-C1 ( Nh eI-DsRed2-F: 5 ′ -GCTAGCATGGCCTCCTCCGAGAAC-3 ′ and DsRed2- Afl II-R: 5 ′ -CTTAAGCTACAGGAACAGGTGGTGGCG-3 ′ ; Clontech, Mountain View, CA, USA) and was cloned into the pGEM-T Easy Vector system (Promega Co., Madison, WI, USA), designated as pGEMT-DsRed2 (pGEMT-eGFP for mKate2).

Techniques: Transformation Assay, Plasmid Preparation, Sequencing, Marker, Fluorescence, Electron Microscopy

Journal: ACS Central Science

Article Title: Edible Matrix Code with Photogenic Silk Proteins

doi: 10.1021/acscentsci.1c01233

Figure Lengend Snippet: Cryptographic key generation of an edible code with three distinct fluorescence colors and digital signature generation with a hash algorithm. (a) Extraction process of digitized output keys from raw fluorescence input images of a representative edible code (7 × 7 matrix). Three different fluorescence images are acquired with an optical set of excitation and emission: eCFP silk code pattern (cyan); λ ex = 415 nm and λ em = 460 nm, eGFP silk code pattern (green); 470 and 525 nm, and mKate2 silk code pattern (red); 530 and 630 nm. Each code pattern generates a 49-bit long binary key K b . The binary keys of three different codes are combined to a digitized key of 147 bits ( K b1 + K b2 + K b3 ). In the case of a 7 × 7 matrix code, the nominal encoding capacity is calculated to be 2 147 (≈ 1.78 × 10 44 ). (b) Convolutional neural network (CNN) architecture for output key extraction of an edible matrix code. A 2D CNN model consists of three convolutional layers and two fully connected layers ( Table S1 ). Batch normalization is applied to each convolutional layer for faster and more stable training. After each batch normalization, the rectified linear unit (ReLU) activation function is applied, and max-pooling is performed. (c) Hashed key generation from the extracted digitized key via a cryptographic hash algorithm (e.g., MD5). Other strong hash functions can be used including SHA-256 and SHA-512. A hashed key can be used for authentication, ensuring key integrity and securing against unauthorized modifications.

Article Snippet: To construct the plasmids, the marker DsRed2 cDNA (eGFP cDNA for mKate2) was amplified by polymerase chain reaction (PCR) using specific primers with Nhe I/ Afl II sites from pDsRed2-C1 ( Nh eI-DsRed2-F: 5 ′ -GCTAGCATGGCCTCCTCCGAGAAC-3 ′ and DsRed2- Afl II-R: 5 ′ -CTTAAGCTACAGGAACAGGTGGTGGCG-3 ′ ; Clontech, Mountain View, CA, USA) and was cloned into the pGEM-T Easy Vector system (Promega Co., Madison, WI, USA), designated as pGEMT-DsRed2 (pGEMT-eGFP for mKate2).

Techniques: Fluorescence, Activation Assay