Team:GreatBay SZ/Hardware

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1. Introduction

In this project, we have designed the Artag system, which can be used for the anti-counterfeiting of art paintings or important documents. The system is divided into three main parts:

  1. Customized DNA barcode design: User-defined DNA information was integrated into the genome of the yeast.
  2. Yeast sporeproduction: Spores were chosen in this project as an ideal carrier for long-time DNA barcode storage and integrated into art-related materials like ink paste.
  3. CRISPR-based nucleic acid detection system: CRISPR-based technology was introduced to detect sequences quickly and efficiently in terms of information decoding.

In order to further promote the Artag system, we designed modular and easy-to-use hardware systems to facilitate more people using the system for their own work. The hardware contains all the tools and reagents needed fro Artag and provides a detailed, streamlined operational guidance for personal customers.

2. Application scenarios

The customers can customise a DNA Barcode containing the user's required information from Artag (assuming that Artag is a commercial company). This Artag will synthesize the Barcode and creates yeast spore ponder with the Barcode information integrated into the genome. The company then provides the user with a hardware device with the customized spores.

Once the user receives the hardware device, he or she can make his or her own inkpaste containing yeast spores and make special seals on his or her artwork.

Once the authenticity of the artwork needs to be verified, the user can directly use the various tools and reagents in the hardware to decode the Bacode on site. The results will be clearly shown on the test strips, which gives clear information to determine the authenticity of the work.

3. Appearance of the hardware

The surface of the hardware has been designed to incorporate elements from Chinese culture. It consists of a kind of wooden box which used to be a device in traditional Chinese culture to collect cosmetics, decorated with artistic paintings and geometric patterns inspired by traditional Chinese culture.

The wooden box structure will be divided into several levels to carry different modular tools and reagents of Artag system, details of which will be presented in the next section.

4. Composition of Hardware

The hardware consists of four Kits. Kit A was for applying engineered spores to the artwork and Kits B-D was for artwork authentication.

Kit A: Sample Preparation Kit — making inkpaste containing spores with DNA barcode integrated

Content ARTAG-SPK(1 prep)
Spores ponder with customized DNA barcode 1 g × 1 tube
DMSO solvent 5 mL × 1 tube
Red Inkpaste 15 g × 1 dish
Seal or Stamp One

* Room Temperature Storage

Kit B: Sampling Kit — sampling on an artwork for analysis

Content ARTAG-SK(50 preps)
Sterile Nylon Swab 50 × 1 bag
Sterile Nylon Solution (0.15 M NaCl +0.1% Tween-20) 50mL × 1 tube
200mM NaOH 10mL × 1 tube
2M HCl 1mL × 1 tube
10x TE Buffer(Tris-HCl 100 mM,EDTA 10 mM,pH 8.0) 1mL × 1 tube
2m Sample Bottle 50 × 1 bag

* Room Temperature Storage

Kit C: RPA Kit — amplifying barcode sequence at room temperature

Content ARTAG-PRAK(48 preps)
A buffer 1.6 mL × 1 tube
B buffer 150 μL × 1 tube
Forward primer 48 μL × 1 tube
Reverse primer 48 μL × 1 tube
ddH2O 144 μL × 1 tube
Reaction tube 48 × 1 bag

* Room Temperature Storage (A & Buffer should be stored in freezer)

Kit D: CRISPR-Cas12a kit — detecting barocde

Content ARTAG-CCK(48 preps)
Nuclease-free water 960 μL × 1 tube
10X NEBuffer 2.1 144 μL × 1 tube
1uM EnGen Lba Cas12a 48 μL × 1 tube
300mM crRNA 144 μL × 1 tube
HEX(Fluorescence) / FAM(test strip) probe 48 μL × 1 tube
Test strip 48 × 1 bga

* Room Temperature Storage (Buffer and Cas12a enzyme should be stored in freezer)

5. Operating instructions for customers

Making inkpaste containing spores with DNA barcode integrated

Materials Needed
Reagent Volume
Spore ponder 60 mg
DMSO 300 μL
Inkpaste 1g

1. Prepare 0.06g dried spore,1g inkpad,30Oul DMSO.

2. Crush the dried spore into powder using mortar and pestle.

3. Dissolve 0.06g dried spore into 300ul DMSO.(it takes very long time to dissolve, it better to put it into shaker or using vortex oscillator).

4. Add dissolved spores into the inkpad and mix them thoroughly.

5. Mark the artwork using a stamp or seal.

The steps to Identify the authenticity of artworks

Step 1: Sampling of paintings with minimal damage using Kit B
Materials Needed
Reagent Volume
Sterile Nylon Swab One
Sterile Nylon Solution (0.15 M NaCl +0.1% Tween-20) \
200mM NaOH 200 μL
2M HCl 20 μL
10x TE Buffer(Tris-HCl 100 mM,EDTA 10 mM,pH 8.0) 20 μL

1. Immerse a sterile nylon swab in a sterile swab solution (0.15 M NaCl + 0.1% Tween-20) and wipe off excess liquid

2. A moistened swab is rubbed over the object for sampling, covering each part of the object surface. Repeat once

3. Hold the tip of the swab in a microcentrifuge tube and apply 200 μL of freshly prepared 200 mM NaOH to the swab.

4. Heat the tube to 95°C for 10 minutes, then neutralise the base with 20 μL of 2 M HCl and add 20 μL of 10x TE buffer (Tris-HCl 100 mM, EDTA 10 mM, pH 8.0).

Step 2: amplification of Barcode using Kit C at room temperature
Materials Needed
Reagent Volume
A buffer 29.4 μL
Forward primer 2 μL
Reverse primer 2 μL
ddH2O and template 14.1 μL
B buffer 2.5 μL
Total 50 μL

1. Take out the required components of the kit 30 minutes in advance, melt at room temperature, shake and mix well.

2. Add 29.4μL A buffer to each dry powder reaction tube (Note: A buffer needs to be completely melted and mixed, otherwise it will affect the experimental effect);

3. Add 2μL forward and 2μL reverse to each reaction tube (primer concentration is 10uM, for multiple reactions, step 1 and step 2 can be mixed and then dispensed into the reaction tube);

4. Add 11.5μL ddH2O and 2uL nucleic acid template to the reaction tube (the volume of nucleic acid template added can be adjusted according to the nucleic acid concentration, and the volume of added ddH2O can be adjusted accordingly, until the total volume of template and ddH2O is 14.1uL);

5. Finally, add 2.5μL of B buffer to the reaction tube and mix it thoroughly (be sure to turn the reaction tube upside down 8-10 times for mixing; for multiple reactions, it is recommended to add B buffer to the inside of the lid of the reaction tube upside down After mixing);

6. After mixing, shake the reaction solution (or quickly centrifuge) to the bottom of the tube, and then immediately put the reaction tube in a constant temperature device for 37-39 incubation for 8 -12 mins;

Step 3: Confirmation of Barcode sequences using Kit D
Materials Needed
Reagent Volumn
Nuclease-free water 19 uL
10X NEBuffer 2.1 3 uL
1uM EnGen Lba Cas12a 1 uL
300mM crRNA 3 uL
HEX(Fluorescence) / FAM(test strip) probe 1 uL
Total Volumn 27 uL

1. Assemble the reaction at room temperature according to the table above

2. Pre-incubate for 10 minutes at 25⁰C.

3. Add 3 µl of 30 nM substrate DNA (30 µl final volume).

4. Mix thoroughly and pulse-spin in a microfuge.

5. Incubate at 37°C for 10 minutes.

6. Incubate at room temperature for 10 minutes.

7. Proceed with analysis.

*Wear a mask during the experiment to avoid RNase contamination.

6. In conclusion

To promote the Artag system, we have designed modular and user-friendly hardware kits. The tools and reagents are assorted into different drawers according to the procedure, together with detailed and streamlined operational guidance, consumers are able to go through the detection procedure step-by-step without technical difficulties.

We hope that this hardware device will help Artag to achieve a wider real-world impact by helping the artists, government staff, and others to preserve the authenticity of their work.