iGEM 2025 — Team shanghai-city

{% extends "layout.html" %}
{% set table\_data = [{
"title": "Overview",
"content": '

The concentrated experimental period was from July 21
to August 31, 2025. After the experiments were completed, the data were organized, and preliminary results were
reported. All these members participated in every aspect of the experiment.

**The daily experimental schedule for the
project is shown in the following table:**

**Table 1. Experimental Schedule for the
Project**

|  |  |  |
| --- | --- | --- |
| **No.** | Date | **Experimental tasks** |
| **1** | 2025/07/21-2025/07/31 | Project Background Learning and Team Icebreaker |
| **2** | Prepare LB medium, amplify genes by PCR, and inoculate culture |
| **3** | Plasmid extraction, linearization of pET-28a plasmid, agarose gel preparation, agarose gel electrophoresis, homologous recombination ligation, heat shock transformation, and transformation plate culture |
| **4** | Monoclonal PCR, shaking culture, agarose gel electrophoresis, preparation of 40% glycerol and kanamycin, strain expansion culture |
| **5** | Strain preservation, plasmid extraction, strain expansion culture, monitoring of OD600 value of bacterial culture, IPTG induction of protein expression |
| **6** | 2025/08/09-2025/08/20 | Collecting bacterial cells, extracting and purifying proteins, preparing SDS-PAGE gels, performing SDS-PAGE gel electrophoresis, staining and destaining gels |
| **7** | Plasmid extraction, linearization of pET-28a plasmid, agarose gel preparation, agarose gel electrophoresis, homologous recombination ligation, heat shock transformation, and transformation plate culture |
| **8** | RPA amplification using different RPA primers, Cas12a cleavage of RPA products, fluorescence detection, feasibility analysis (fluorescence method and test strip method), optimization of Cas12a cleavage time, and gradient dilution of plasmids |
| **9** | Optimal RPA primers for RPA amplification, Cas12a cleavage of RPA products, fluorescence detection, and test strip analysis: sensitivity analysis + random sample analysis |
| **10** | 2025/08/29-2025/09/10 | Organize data and summarize experiments |
| **11** | Wiki writing |

**The specific experimental records are as
follows:**

'
},{
"title": "Date: July, 2025",
"content": '

1. **Experimental Contents**
   1. **Prepare LB medium**

      Preparation of LB Medium: According to the LB
      medium
      formula, 10g of tryptone, 5g of yeast extract, and 10g of sodium chloride (NaCl) are required per 1L. For
      solid
      medium, an additional 15 - 20g/L of agar powder is needed. Calculate proportionally according to this formula:
      To
      prepare 200 mL of liquid medium and 200 mL of solid medium, 2g of tryptone, 1g of yeast extract, and 2g of
      sodium
      chloride are required. For the solid medium, an additional 3 - 4g of agar powder is needed. Add and mix all
      the
      required raw materials, seal the bottle mouth, and sterilize at high temperature and high pressure, 121
      degrees
      Celsius for 15-20 min. After cooling to 60℃, Kanamycin (Kana) was added to the culture medium until the final
      concentration of Kana was 50 mg/L. Specifically, the concentration of the Kana solution stock solution is 50
      mg/ mL,
      and 100 microliters (μL) of the Kana solution stock solution should be added to every 100 mL of the culture
      medium.
   2. **PCR amplification of genes**

      A-Y: COL7A1 c.520G

      A-T: COL7A1 c.520G>A

      B-Y: COL7A1 c.6745C

      B-T: COL7A1 c.6745C>T

      Through the PCR gene amplification
      technology, the
      required DNA fragments. A-Y and B-Y are the wild sequences, corresponding to c.520G and c.6745C respectively.
      A-T and
      B-T are the mutant sequences, corresponding to c.520G>A and c.6745C>T. N is the control group. For B-Y,
      add 50
      μL of ddH₂O, 2 μL of Primer-1, 25 μL of 2Taq Master Mix, and 1 μL of Template DNA; for B-T, add 50 μL of
      ddH₂O, 2 μL
      of Primer-2, 25 μL of 2Taq Master Mix, and 1 μL of Template DNA; for N, add 50 μL of ddH₂O, 2 μL of Primer-2,
      and 25
      μL of 2×Taq Master Mix. Put it into a PCR instrument and set the following reaction program: keep at 95
      degrees
      Celsius for 1 minute, then enter the cycling stage: 95 degrees Celsius for 30 seconds, 56 degrees Celsius for
      30
      seconds, 72 degrees Celsius for 4 minutes,
      cycGGGGCTGCAATTCTCCATGTGGCTGACCATGTCTTCCTGCCCCAGCTGGCCCGACCTGGTGTCCCCAAGGTCTGCATCCTGATCACAGACGGGAAGTCCCAGGACCTGGTGGACACAGCTGCCCAAAGGCTGAAGGGGCAGGGGGTCAAGCTAle
      30 - 35 times. After the cycle ends, finally keep at 72 degrees
      Celsius for 5 minutes, and then keep at 12 degrees Celsius.

      The gene sequences are as follows:

      **AsCpf1 sequence:**

      ATGACCCAATTTGAAGGTTTTACCAATTTATACCAAGTTTCGAAGACCCTTCGTTTTGAACTGATTCCCCAAGGAAAAACACTCAAACATATCCAGGAGCAAGGGTTCATTGAGGAGGATAAAGCTCGCAATGACCATTACAAAGAGTTAAAACCAATCATTGACCGCATCTATAAGACTTATGCTGATCAATGTCTCCAACTGGTACAGCTTGACTGGGAGAATCTATCTGCAGCCATAGACTCCTATCGTAAGGAAAAAACCGAAGAAACACGAAATGCGCTGATTGAGGAGCAAGCAACATATAGAAATGCGATTCATGACTACTTTATAGGTCGGACGGATAATCTGACAGATGCCATAAATAAGCGCCATGCTGAAATCTATAAAGGACTTTTTAAAGCTGAACTTTTCAATGGAAAAGTTTTAAAGCAATTAGGGACCGTAACCACGACAGAACATGAAAATGCTCTACTCCGTTCGTTTGACAAATTTACGACCTATTTTTCCGGCTTTTATGAAAACCGAAAAAATGTCTTTAGCGCTGAAGATATCAGCACGGCAATTCCCCATCGAATCGTCCAGGACAATTTCCCTAAATTTAAGGAAAACTGCCATATTTTTACAAGATTGATAACCGCAGTTCCTTCTTTGCGGGAGCATTTTGAAAATGTCAAAAAGGCCATTGGAATCTTTGTTAGTACGTCTATTGAAGAAGTCTTTTCCTTTCCCTTTTATAATCAACTTCTAACCCAAACGCAAATTGATCTTTATAATCAACTTCTCGGCGGCATATCTAGGGAAGCAGGCACAGAAAAAATCAAGGGACTTAATGAAGTTCTCAATCTGGCTATCCAAAAAAATGATGAAACAGCCCATATAATCGCGTCCCTGCCGCATCGTTTTATTCCTCTTTTTAAACAAATTCTTTCCGATCGAAATACGTTATCCTTTATTTTGGAAGAATTCAAAAGCGATGAGGAAGTCATCCAATCCTTCTGCAAATATAAAACCCTCTTGAGAAACGAAAATGTACTGGAGACTGCAGAAGCCCTTTTCAATGAATTAAATTCCATTGATTTGACTCATATCTTTATTTCCCATAAAAAGTTAGAAACCATCTCTTCAGCGCTTTGTGACCATTGGGATACCTTGCGCAATGCACTTTACGAAAGACGGATTTCTGAACTCACTGGCAAAATAACAAAAAGTGCCAAAGAAAAAGTTCAAAGGTCATTAAAACATGAGGATATAAATCTCCAAGAAATTATTTCTGCTGCAGGAAAAGAACTATCAGAAGCATTCAAACAAAAAACAAGTGAAATTCTTTCCCATGCCCATGCTGCACTTGACCAGCCTCTTCCCACAACATTAAAAAAACAGGAAGAAAAAGAAATCCTCAAATCACAGCTCGATTCGCTTTTAGGCCTTTATCATCTTCTTGATTGGTTTGCTGTCGATGAAAGCAATGAAGTCGACCCAGAATTCTCAGCACGGCTGACAGGCATTAAACTAGAAATGGAACCAAGCCTTTCGTTTTATAATAAAGCAAGAAATTATGCGACAAAAAAGCCCTATTCGGTGGAAAAATTTAAATTGAATTTTCAAATGCCAACCCTTGCCTCTGGTTGGGATGTCAATAAAGAAAAAAATAATGGAGCTATTTTATTCGTAAAAAATGGTCTCTATTACCTTGGTATCATGCCTAAACAGAAGGGGCGCTATAAAGCCCTGTCTTTTGAGCCGACAGAAAAAACATCAGAAGGATTCGATAAGATGTACTATGACTACTTCCCAGATGCCGCAAAAATGATTCCTAAGTGTTCCACTCAGCTAAAGGCTGTAACCGCTCATTTTCAAACTCATACCACCCCCATTCTTCTCTCAAATAATTTCATTGAACCTCTTGAAATCACAAAAGAAATTTATGACCTGAACAATCCTGAAAAGGAGCCTAAAAAGTTTCAAACGGCTTATGCAAAGAAGACAGGCGATCAAAAAGGCTATAGAGAAGCGCTTTGCAAATGGATTGACTTTACGCGGGATTTTCTCTCTAAATATACGAAAACAACTTCAATCGATTTATCTTCACTCCGCCCTTCTTCGCAATATAAAGATTTAGGGGAATATTACGCCGAACTGAATCCGCTTCTCTATCATATCTCCTTCCAACGAATTGCTGAAAAGGAAATCATGGATGCTGTAGAAACGGGAAAATTGTATCTGTTCCAAATCTACAATAAGGATTTTGCGAAGGGCCATCACGGGAAACCAAATCTCCACACCCTGTATTGGACAGGTCTCTTCAGTCCTGAAAACCTTGCGAAAACCAGCATCAAACTTAATGGTCAAGCAGAATTGTTCTATCGACCTAAAAGCCGCATGAAGCGGATGGCCCATCGTCTTGGGGAAAAAATGCTGAACAAAAAACTAAAGGACCAGAAGACACCGATTCCAGATACCCTCTACCAAGAACTGTACGATTATGTCAACCACCGGCTAAGCCATGATCTTTCCGATGAAGCAAGGGCCCTGCTTCCAAATGTTATCACCAAAGAAGTCTCCCATGAAATTATAAAGGATCGGCGGTTTACTTCCGATAAATTTTTCTTCCATGTTCCCATTACACTGAATTATCAAGCAGCCAATAGTCCCAGTAAATTCAACCAGCGTGTCAATGCCTACCTTAAGGAGCATCCGGAAACGCCCATCATTGGTATCGATCGTGGAGAACGCAATCTAATCTATATTACCGTCATTGACAGTACTGGGAAAATTTTGGAGCAGCGTTCCCTGAATACCATCCAGCAATTTGACTACCAAAAAAAATTGGACAACAGGGAAAAAGAGCGTGTTGCCGCCCGTCAAGCCTGGTCCGTCGTCGGAACGATCAAAGACCTTAAACAAGGCTACTTGTCACAGGTCATCCATGAAATTGTAGACCTGATGATTCATTACCAAGCTGTTGTCGTCCTTGAAAACCTCAACTTCGGATTTAAATCAAAACGGACAGGCATTGCCGAAAAAGCAGTCTACCAACAATTTGAAAAGATGCTAATAGATAAACTCAACTGTTTGGTTCTCAAAGATTATCCTGCTGAGAAAGTGGGAGGCGTCTTAAACCCGTATCAACTTACAGATCAGTTCACGAGCTTTGCAAAAATGGGCACGCAAAGCGGCTTCCTTTTCTATGTACCGGCCCCTTATACCTCAAAGATTGATCCCCTGACTGGTTTTGTCGATCCCTTTGTATGGAAGACCATTAAAAATCATGAAAGTCGGAAGCATTTCCTAGAAGGATTTGATTTCCTGCATTATGATGTCAAAACAGGTGATTTTATCCTCCATTTTAAAATGAATCGGAATCTCTCTTTCCAGAGAGGGCTTCCTGGCTTCATGCCAGCTTGGGATATTGTTTTCGAAAAGAATGAAACCCAATTTGATGCAAAAGGGACGCCCTTCATTGCAGGAAAACGAATTGTTCCTGTAATCGAAAATCATCGTTTTACGGGTCGTTACAGAGACCTCTATCCCGCTAATGAACTCATTGCCCTTCTGGAAGAAAAAGGCATTGTCTTTAGAGACGGAAGTAATATATTACCCAAACTTTTAGAAAATGATGATTCTCATGCAATTGATACGATGGTCGCCTTGATTCGCAGTGTACTCCAAATGAGAAACAGCAATGCCGCAACGGGGGAAGACTACATCAACTCTCCCGTTAGGGATCTGAACGGGGTGTGTTTCGACAGTCGATTCCAAAATCCAGAATGGCCAATGGATGCGGATGCCAACGGAGCTTATCATATTGCCTTAAAAGGGCAGCTTCTTCTGAACCACCTCAAAGAAAGCAAAGATCTGAAATTACAAAACGGCATCAGCAACCAAGATTGGCTGGCCTACATTCAGGAACTGAGAAACTGA

      **A-Y sequence:**

      GGGGCTGCAATTCTCCATGTGGCTGACCATGTCTTCCTGCCCCAGCTGGCCCGACCTGGTGTCCCCAAGGTCTGCATCCTGATCACAGACGGGAAGTCCCAGGACCTGGTGGACACAGCTGCCCAAAGGCTGAAGGGGCAGGGGGTCAAGCTATTTGCTGTGGGGATCAAGAATGCTGACCCTGAGGAGCTGAAGCGAGTTGCCTCACAGCCCACCAGTGACTTCTTCTTCTTCGTCAATGACTTCAGCATCTTGAGGACACTACTGCCCCTCGTTTCCCGGAGAGTGTGCACGACTGCTGGTGGCGTGCCTGTGACCCGACCTCCGGATGACTCGACCTCTGCTCCACG

      **A-T sequence:**

      GGGGCTGCAATTCTCCATGTGGCTGACCATGTCTTCCTGCCCCAGCTGGCCCGACCTGGTGTCCCCAAGGTCTGCATCCTGATCACAGACGGGAAGTCCCAGGACCTGGTGGACACAGCTGCCCAAAGGCTGAAGGGGCAGGGGGTCAAGCTATTTGCTGTGAGGATCAAGAATGCTGACCCTGAGGAGCTGAAGCGAGTTGCCTCACAGCCCACCAGTGACTTCTTCTTCTTCGTCAATGACTTCAGCATCTTGAGGACACTACTGCCCCTCGTTTCCCGGAGAGTGTGCACGACTGCTGGTGGCGTGCCTGTGACCCGACCTCCGGATGACTCGACCTCTGCTCCACG

      **B-Y sequence:**

      CTTGCTGGCCCTGCAGGACCCCAAGGACCTTCTGGCCTGAAGGGGGAGCCTGGAGAGACAGGACCTCCAGGACGGGGCCTGACTGGACCTACTGGAGCTGTGGGACTTCCTGGACCCCCCGGCCCTTCAGGCCTTGTGGGTCCACAGGGGTCTCCAGGTTTGCCTGGACAAGTGGGGGAGACAGGGAAGCCGGGAGCCCCAGGTCGAGATGGTGCCAGTGGAAAAGATGGAGACAGAGGGAGCCCTGGTGTGCCAGGGTCACCAGGTCTGCCTGGCCCTGTCGGACCTAAAGGAGAAC

      **B-T sequence:**

      CTTGCTGGCCCTGCAGGACCCCAAGGACCTTCTGGCCTGAAGGGGGAGCCTGGAGAGACAGGACCTCCAGGACGGGGCCTGACTGGACCTACTGGAGCTGTGGGACTTCCTGGACCCCCCGGCCCTTCAGGCCTTGTGGGTCCACAGGGGTCTCCAGGTTTGCCTGGATAAGTGGGGGAGACAGGGAAGCCGGGAGCCCCAGGTCGAGATGGTGCCAGTGGAAAAGATGGAGACAGAGGGAGCCCTGGTGTGCCAGGGTCACCAGGTCTGCCTGGCCCTGTCGGACCTAAAGGAGAAC

      The primer sequence is as follows:

      **Table 2. PCR Primer
      information**

      |  |  |  |  |
      | --- | --- | --- | --- |
      | **Gene** | **Primer Name** | **Sequence (5’-3’)** | **Amplicon size** |
      | ASCpf1 | PCR-ASCpf1-F | AGCAAATGGGTCGCGGATCCATGACTCAGTTCGAA | 3936 bp |
      | PCR-AsCpf1-R | TGTCGACGGAGCTCGAATTCTTAGTTACGCAGTTC |
      | c .520G | PCR-c .520G-F | CAAATGGGTCGCGGATCCGGGGCTGCAATTCTCCA | 362 bp |
      | PCR-c .520G-R | CTTGTCGACGGAGCTCGAATTCCGTGGAGCAGAGG |
      | c .520G>A | PCR-c .520G>A-F | AAATGGGTCGCGGATCCGGGGCTGCAATTCTCCAT | 362 bp |
      | PCR-c .520G>A-R | TGTCGACGGAGCTCGAATTCCGTGGAGCAGAGGTC |
      | c.6745C | PCR-c.6745C-F | GCAAATGGGTCGCGGATCCCTTGCTGGCCCTGCAG | 310 bp |
      | PCR-c.6745C-R | CTTGTCGACGGAGCTCGAATTCGTTCTCCTTTAGG |
      | c.6745C>T | PCR-c.6745C>T-F | AAATGGGTCGCGGATCCCTTGCTGGCCCTGCAGGA | 310 bp |
      | PCR-c.6745C>T-R | GCTTGTCGACGGAGCTCGAATTCGTTCTCCTTTAG |
   3. **Inoculation of bacterial culture**

      Add 3 mL of LB liquid culture medium and 3 μL
      of 50
      mg/ mL Kana resistance solution to a sterile test tube, mix well, and inoculum 3 μL of TOP10 bacterial liquid
      containing pET28a plasmid. Then, incubate the inoculation liquid in a 37 ℃ shaker at 220 rpm for 12-16 hours.

**Date: July, 2025**

1. **Experimental Contents**
   1. **Plasmid extraction**

      **Step 1:**
      Scientists took 3 mL of the
      fifteen-hour cultured bacterial liquid and added it to a centrifuge tube. Then, it is centrifuged at 10,000
      rpm
      (11,500×g) for 1 minute. The culture medium was then discarded as much as possible.

      **Step 2:**
      Scientists then added 250 μL
      of Buffer P1that contains RNase A, to the centrifuge tube with the bacterial pellet, and mixed well with a
      vortex
      oscillator. This process resuspended the bacteria.

      **Step 3:** Then,
      250 μL of Buffer P2 is
      added to the centrifuge tube and is mixed with gentle inversion 8-10 times to lyse the bacteria fully.

      **Step 4:** Then,
      350 μL of Buffer P3 is
      added to the centrifuge tube, and is mixed with gentle inversion 8-10 times to neutralize Buffer P2
      thoroughly. At
      this point, there should be white flocculent precipitation. The tube is then centrifuged at 12,000 rpm
      (13,400×g) for
      10 minutes.

      **Step 5:** Then,
      Scientists place the
      FastPure DNA Mini Columns in a 2 mL collection tube. The supernatant from the previous step is then
      transferred to the
      adsorption column with a pipette, and later centrifuged at 12,000 rpm (13,400×g) for 45 seconds. The waste
      liquid in
      the collection tube is then discarded. This process is meant for column loading and plasmid binding.

      **Step 6:**
      Scientists then added 500 μL
      of Buffer PW1 to the adsorption column, which was centrifuged at 12,000 rpm (13,400×g) for 45 seconds. The
      waste
      liquid is then discarded. This process is to wash off the protein.

      **Step 7:** 600
      μL of Buffer PW2 diluted
      with anhydrous ethanol is then added to the adsorption column. Centrifuged at 12,000 rpm (13,400×g) for 45
      seconds.
      The waste liquid is then discarded.

      **Step 8:** The
      process above is
      repeated twice, washing off the salt ions.

      **Step 9:** The
      adsorption column is
      then placed back in the collection tube and centrifuged at 12,000 rpm (13,400×g) for 1 minute to dry the
      adsorption
      column, removing the rest of the washing solution in the adsorption column.

      **Step 10:** The
      adsorption column is
      then placed in a new sterile 1.5 mL centrifuge tube. Then, 100 μL of Elution Buffer is added to the center of
      the
      membrane of the adsorption column. The column is then placed at room temperature for 2 minutes, and
      centrifuged at
      12,000 rpm (13,400×g) for 1 minute to elute the DNA. This process washes off the protein.

      **Step 11:** The
      adsorption column is
      then discarded, and the DNA product is stored at -20℃ to prevent DNA degradation.
   2. **Concentration testing using Nanodrop**

      The concentrations of the plasmid samples
      were
      measured using Nanodrop.
   3. **Linearization of the pET-28a plasmid**

      **Objective:**

      Digest 1 μL of pET-28a plasmid using the
      restriction
      endonucleases EcoRI and BamHI.

      **Setup:**

      A total of three groups were prepared, with
      each
      group containing two reaction systems (six systems in total). Each reaction system had a final volume of 50
      μL,
      consisting of the following components:

      * 10 μL of pET-28a plasmid
      * 1 μL of EcoRI
      * 1 μL of BamHI
      * 5 μL of CUM Smart Buffer
      * 33 μL of ddH₂O (double-distilled water)

      **Procedure:**

      The reaction mixtures were incubated at 37 ℃
      for 10
      minutes to complete the digestion and linearize the plasmid.
   4. **Preparation of Agarose Gel**

      **Materials:**

      1**×**TAE buffer, agarose
      powder.

      **Equipment:**

      Gel casting tray, gel comb, electronic
      balance,
      graduated cylinder, Erlenmeyer flask, micropipette **Procedure:**

      1. **Calculation:**

         For this experiment, a small gel casting
         tray is
         used, requiring 30 mL of TAE buffer and 0.45 g of agarose powder.
      2. **Measurement:**

         Weigh approximately 0.45 g of agarose
         powder using an
         electronic balance. Measure 30 mL of TAE buffer using a graduated cylinder and micropipette.
      3. **Mixing:**

         Add the agarose powder and TAE buffer
         into an
         appropriately sized Erlenmeyer flask. Gently swirl the flask until the agarose powder partially dissolves.
         Then, place
         the flask in a heating device (microwave) and heat until all agarose particles are completely dissolved.
         After
         heating, the solution should appear clear.
      4. **Staining:**

         Add 2 µL of nucleic acid stain to the
         solution and
         gently swirl until the solution shows a light pink color.
      5. **Gel Casting:**

         Slowly pour the prepared solution
         (agarose solution
         with nucleic acid stain) into the gel casting tray to avoid bubble formation, which can interfere with
         observation.
         Let the solution sit undisturbed until it solidifies into a gel.
   5. **Agarose gel electrophoresis**

      Agarose gel electrophoresis was used to
      analyze PCR
      products and restriction enzyme digestion products. Scientists first inserted 5 μL of each product into
      designated
      columns in order as follows.

      M: DNA Marker

      M A-Y A-Y A-Y N M A-T A-T A-T N M

      M B-Y B-Y B-Y N M B-T B-T B-T N M

      M AsCpf1 AsCpf1 AsCpf1 N M
   6. **Purify PCR product and plasmid enzyme-digested product using Vazyme Gel DNA Extraction Mini kit**

      **Process:**

      1. Transfer 50 μL enzyme-digested product and 100 μL PCR product into two different adsorption columns
         respectively, and then 12,000 rpm (13,800 ×g) centrifuge 40 seconds. DNA is adsorbed on the membrane of the
         adsorption column.
      2. Discard the filtrate and place the adsorption column in the collection tube. Add 300 μL of Buffer GDP to
         the
         adsorption column. Let it stand for 1 minute, and then 12,000 rpm (13,800 ×g) centrifuge for 40 seconds.
      3. Discard the filtrate and place the adsorption column in the collection tube. Add 700 μL Buffer GW
         (anhydrous
         ethanol has been added) to the adsorption column. Cover the lid, invert the collection tube 3 times to help
         thoroughly rinse off the salt adhering to the pipe wall, and then 12,000 rpm (13,800 ×g) centrifuge 40
         seconds.
      4. Repeat step 3 to make sure the salt is completely removed.
      5. Discard the filtrate and place the adsorption column in the collection tube. 12,000 rpm (13,800 ×g)
         centrifuge
         for 2 minutes.
      6. Place the adsorption column in 1.5 mL sterilized centrifuge tubes. Add 30 μL ddH2O into the
         middle
         of the adsorption column, place it for 2 minutes, and then 12,000 rpm (13,800 ×g) centrifuge 1 minute.
         Discard
         the adsorption column, detect the concentration of the products, and store the DNA at -20℃.
   7. **Homologous Recombination-based Ligation**

      **Goal:**

      Ligation of the linearized pET28a fragment
      and the
      gene fragment

      **Procedure:**

      Using the Vazyme C115 Clon Express Ultra One
      Step
      Cloning kit to achieve homologous recombination ligation.

      **Calculation of 0.03pmol of Linearized
      vector
      pET-28a（5353bp):**

      0.02×5353=106.5ng

      **The volume of the linearized vector
      (10
      µg/µL) to be added should be:**

      106.5÷10≈10µL

      **Calculation of insert
      fragments：**

      AsCpf1: n=0.04×bp=0.04×3936=157.44ng

      v=n/c=157.44/180≈1µL

      A-Y：n=0.04×362=14.48µg

      v=n/c=14.48≈1µL

      A-T：n=0.04×362=14.48µg

      v=n/c=14.48≈1µL

      B-Y：n=0.04×310=12.4µg

      v=n/c=12.4/77.15≈1µL

      B-T：n=0.04×310=12.4µg

      v=n/c=12.4/79.05≈1µL

      1µL of each of the five recombinant products.

      **Table 3. Homologous recombination
      ligation
      system**

      |  |  |  |
      | --- | --- | --- |
      | **Component** | **×1 (Total 10µL)** | **Amount Added (µL)** |
      | 2×Clon Express mix | 5µL | 5 |
      | Linearized vector pET28a | 0.03pmol=0.02×bp(µg) | 4 |
      | Insert fragments | 0.006pmol=0.04×bp(ng) | 1 |
      | ddH2O | adjust the volume to 10µL | 0 |

      **Heat the 10 µL recombinant product at
      50 ℃
      for 20 min (to bind the insert fragments onto pET-28a).**

      Then, AsCpf1, A-Y, A-T, B-Y, and B-T
      fragments were
      ligated into the linearized pET28a vector, respectively, forming new recombinant circular plasmids
      pET28a-AsCpf1,
      pET28a-A-Y, pET28a-A-T, pET28a-B-Y, and pET28a-B-T.
   8. **Pouring LB Plates**

      After the LB solid culture medium has cooled
      to
      approximately 60 ℃, add 200 µL of 50 mg/ mL Kana stock solution into 200 mL of LB solid culture medium, mix
      well, and
      pour into sterile plates until the surface is fully covered. A total of 12 plates were poured.
   9. **Heat-Shock Transformation**

      **Goal：**

      Insert the recombinant plasmid pET28a-AsCpf1
      into
      BL21 cells to express the target protein.

      Insert the recombinant plasmids
      pET28a-AsCpf1,
      pET28a-A-Y, pET28a-A-T, pET28a-B-Y, and pET28a-B-T into DH5α cells to clone more DNA.

      1. Take 10 µL of the AsCpf1 recombinant product and add it to 100 µL of DH5α competent cells; then take
         another 10 µL
         of the AsCpf1 recombinant product and add it to 100 µL of BL21 competent cells.
      2. Incubate on ice for 30 min.
      3. Perform heat shock in a 42 ℃ water bath for 45 sec.
      4. Place on ice for 1 min.
      5. Add 900 µL of LB medium.
      6. Spread the cultures onto plates—distribute the two 100 µL bacterial suspensions onto 2 plates each using
         the
         spread plate method.
      7. Incubate the cultures at 37 ℃ for 12–16 h.
2. **Results**
   1. **Concentration testing using Nanodrop**

      Gene AsCpf1 has a concentration of 19.6
      ng/μL.
      plasmid pET-28a has a concentration of 5.8 ng/μL.

      The concentrations of the plasmid samples
      were
      measured. to be 36.2 ng/μL, 37.0 ng/μL, 66.8 ng/μL, 69.5 ng/μL, 32.6 ng/μL, and 36.2 ng/μL, respectively.
   2. **Gene amplification electrophoresis results**

      **AsCpf1 gene amplification
      electrophoresis
      results:**

      ![](https://static.igem.wiki/teams/6004/notebook/1.webp)

      In the electrophoresis gel, a distinct band
      around
      3936 bp was observed, indicating the successful amplification of the AsCpf1 gene.

      **A-Y gene amplification
      electrophoresis
      results:**

      ![](https://static.igem.wiki/teams/6004/notebook/2.webp)

      A distinct band around 362 bp was observed,
      indicating the successful amplification of the A-Y gene.

      **A-T gene amplification
      electrophoresis
      results:**

      ![](https://static.igem.wiki/teams/6004/notebook/3.webp)

      A distinct band around 362 bp was observed,
      indicating the successful amplification of the A-T gene.

      **B-Y gene amplification
      electrophoresis
      results:**

      ![](https://static.igem.wiki/teams/6004/notebook/26.webp)

      A distinct band around 310 bp was observed,
      indicating the successful amplification of the B-Y gene.

      **B-T gene amplification
      electrophoresis
      results:**

      ![](https://static.igem.wiki/teams/6004/notebook/4.webp)

      A distinct band around 310 bp was observed,
      indicating the successful amplification of the B-T gene.
   3. **Plasmid linearization results**

      ![](https://static.igem.wiki/teams/6004/notebook/5.webp)

      The recombinant pET28a-AsCpf1, pET28a-A-Y,
      pET28a-A-T, pET28a-B-Y, and pET28a-B-T plasmids, along with the linearized pET-28a fragment, and the AsCpf1,
      A-Y, A-T,
      B-Y, and B-T genes were electrophoresed on the same agarose gel. From the electrophoresis results, it is
      evident that
      the pET-28a vector was successfully linearized. And all genes were successfully amplified.

**Date: July, 2025**

**Goal:**

1. Observe the growth of bacteria on the transformation plates
2. Verify whether the bacteria strain contains the target plasmid
3. Inoculation the bacterial solution
4. Preparation of agarose gel
5. Analysis of colony identification results by agarose gel electrophoresis
6. Prepare LB medium
7. Prepare 200 mL of glycerol with a concentration of 40% (Prevent bacteria from being pierced and killed by
   ice
   during refrigeration.)
8. Prepare 5 mL Kana⁺ at a concentration of 50 mg / mL (Make the required plasmid purer)
9. Picking bacteria and Shaking bacteria. (Obtain single colonies and expand culture)

1. **Experimental Contents**
   1. **Observe bacterial culture plates**

      Observe the growth of bacteria on the
      transformation
      plates and take photos to record them.
   2. **Monoclonal PCR**

      Use a sterilized 10 μL pipette to pick a
      single
      colony and transfer it onto the wall of the PCR tube as the DNA template. Pick four A-T, A-Y, B-T, and B-Y
      genes and
      six AsCpf1-DH5α and AsCpf1-BL21 genes. Mix with a vortex mixer, then centrifuge with a centrifuge. Make eight
      PCRs
      according to the formula of 12.5 μL 2×Taq Master Mix, 1 μL forward primer, 1 μL reverse primer, 10.5 μL
      double-distilled water, and DNA template. Heat them at a temperature of 95 degrees Celsius for 3 min and 95
      degrees
      Celsius for 30 s, 56 degrees Celsius for 30 s, 72 degrees Celsius for 4 min, these three steps repeat for 35
      times as
      a cycle, and then 72 degrees Celsius for 5 min and 12 degrees Celsius in an infinite loop.
   3. **Single colony culture**

      Use the same pipette tip used to pick up the
      single
      colony on the plate into 3 mL of LB liquid culture medium (kana, 50 mg/L) and make the same mark as the PCR
      tube.
      Place the inoculated tube in a 37℃ shaker for 12-16 hours (220 rpm).
   4. **Preparation of agarose gel**

      Add agarose and TAE buffer to the flask and
      mix well.
      Boil in a microwave oven and add dye. Mix well to avoid bubbles. Pour into the gelatin plate with the plate
      and insert
      the comb.

      **Table 4. Agarose gel
      formulation**

      |  |  |  |  |
      | --- | --- | --- | --- |
      |  | **small gel** | **medium gel** | **big gel** |
      | agarose | 0.45g | 075g | 1.5g |
      | TAE buffer | 30mL | 50mL | 100mL |
      | dye | 2μL | 4μL | 8μL |
   5. **PCR gel electrophoresis**

      To identify whether there is the target gene
      in the
      bacteria (containing inserted fragments), agarose gel electrophoresis is used to analyze the results of colony
      identification. For A and B, first add Marker, then add wild type, Marker after, then mutant type, finally
      Marker. Put
      the gel into the electrophoresis apparatus for electrophoresis for around 15 min. After it\'s over, get the
      imaging
      system to observe the results.
   6. **Preparation of culture medium**

      For inoculating the bacterial solution,
      cultivate a
      large number of pET-28a-AsCpf1-BL21 bacteria. To prepare 200 mL LB liquid medium, it is required 200 mL dd
      H2O, 2 g tryptone, 2.5 g yeast extract, 2 g NaCl. After preparation, displace the liquid into the
      place at
      121 ℃ for 15-20 min (sterilization).
   7. **Prepare 40% glycerol**

      Prepare 200 mL of glycerin with a
      concentration of
      40%: 80 mL glycerol plus 120 mL of ddH2O, then high-temperature and high-pressure sterilization
      (121 ℃, 20
      min).
   8. **Prepare Kana⁺**

      Prepare Kana⁺ at a concentration of 50 mg
      /mL: Each
      group prepares 5 mL kanamycin sulfate solution, and the concentration should be 50 mg/mL, so each group weighs
      250 mg
      of kanamycin sulfate. Add 250 mg kanamycin sulfate powder into 5 mL ddH2O. When the powder is
      dissolved in
      5 mL, mix it by vortex, then use a syringe and a 0.22 micrometer filter membrane to filter for sterilization,
      and
      collect the filtrate. Make proper marks for subsequent use. (Added into LB medium)
   9. **Picking and shaking bacteria:**

      Inoculating bacterial liquid. Use the same
      pipette
      tip used for picking bacteria in the plate medium to pipette into 3 mL of LB liquid medium (Kana⁺, 50 mg/L),
      and mark
      it with the same mark as the corresponding PCR tube. Incubate the inoculated test tube on a shaker at 37 ℃ for
      12–16 h
      (220 rpm). pET-28a-AsCpf1-BL21, 4 tubes (For protein expression). pET-28a-AsCpf1-DH5α 2 tubes each (For
      preservation
      and plasmid extraction). pET-28a-A-Y-DH5α, pET-28a-A-T-DH5α, 2 tubes each (For preservation and plasmid
      extraction).
      pET-28a-B-Y-DH5α, pET-28a-B-T-DH5α (For preservation and plasmid extraction). Pick 2 tubes of AsCpf1 - BL21
      bacteria
      for each group.
2. **Results**
   ![](https://static.igem.wiki/teams/6004/notebook/6.webp)

   Bacteria grow well on transformation plates, with
   single colonies appearing. All recombinant plasmids were successfully transformed.

**Date: July, 2025**

1. **Experimental Contents**
   1. **Observation of the growth of the bacterial cultures**
   2. **Bacterial culture preservation**

      500 µL of bacterial culture and 500 µL of 40%
      glycerol are added to a sterile centrifuge tube, mixed thoroughly, and stored at −80 ℃. Two tubes of each
      pET28a-AsCpf1, pET28a-A-Y, pET28a-A-T, pET28a-B-Y, and pET28a-B-T. Four tubes of AsCpf1-DH52 and AsCpf1-BL21.
   3. **Extraction of recombinant plasmids**

      The aim was to extract plasmid DNA from E.
      coli:
      pET28a-AsCpf1, pET28a-A-Y, pET28a-A-T, pET28a-B-Y, and pET28a-B-T. The following process was repeated for all
      five
      groups.

      **Step 1:** 3 mL of the 12-hour
      cultured
      bacterial liquid was added to a centrifuge tube. The sample was centrifuged at 10,000 rpm (11,500
      **×**g)
      for 1 minute. The culture medium was then discarded as much as possible.

      **Step 2:** 250 μL of Buffer P1
      (containing RNase A) was added to the centrifuge tube containing the bacterial pellet, and mixed well using a
      vortex
      mixer. This step resuspended the bacteria.

      **Step 3:** 250 μL of Buffer P2
      was
      added to the centrifuge tube and mixed gently by inverting the tube 8–10 times to fully lyse the bacteria.

      **Step 4:** 350 μL of Buffer P3
      was
      added to the centrifuge tube and gently inverted 8–10 times to neutralize Buffer P2. White flocculent
      precipitation
      should appear. The tube was then centrifuged at 12,000 rpm (13,400 **×**g) for 10 minutes.

      **Step 5:** The supernatant from
      the
      previous step was transferred to a FastPure DNA Mini Column placed in a 2 ml collection tube. The column was
      centrifuged at 12,000 rpm (13,400 **×**g) for 45 seconds. The waste liquid in the collection tube
      was
      discarded. This step is for column loading and plasmid binding.

      **Step 6:** 500 μL of Buffer PW1
      was
      added to the adsorption column, and the column was centrifuged at 12,000 rpm (13,400 **×**g) for
      45
      seconds. The waste liquid was discarded. This step washes off proteins.

      **Step 7:** 600 μL of Buffer PW2
      (diluted with anhydrous ethanol) was added to the adsorption column, and the column was centrifuged at 12,000
      rpm
      (13,400 **×**g) for 45 seconds. The waste liquid was discarded.

      **Step 8:** The washing process
      from
      Step 7 was repeated twice to wash off salt ions.

      **Step 9:** The adsorption
      column was
      placed back in the collection tube and centrifuged at 12,000 rpm (13,400 **×**g) for 1 minute to
      dry the
      column, removing any remaining washing solution.

      **Step 10:** The adsorption
      column was
      placed in a new sterile 1.5 ml centrifuge tube. 40 μL of Elution Buffer was added to the center of the
      membrane in the
      adsorption column. The column was left at room temperature for 2 minutes and then centrifuged at 12,000 rpm
      (13,400
      **×**g) for 1 minute to elute the plasmid DNA.

      **Step 11:** The adsorption
      column was
      discarded, and the extracted plasmid DNA was stored at -20 ℃ to prevent degradation.

      This process successfully extracted plasmid
      DNA for
      further analysis and experiments.
   4. **Preparation of LB Liquid Medium**

      **Objective:**

      To provide competent cells with an optimal
      growth
      environment and the necessary nutrients.

      **Step 1:** Eight tubes of 20 mL
      LB
      liquid medium (containing 50 mg/L kanamycin) were prepared.

      **Step 2:** Using the formula n
      = c × v
      and c₁·v₁ = c₂·v₂, the required volume of kanamycin stock solution was calculated: v1=(c2\*v2)/c1=(50
      mg/L\*20\*10^-3
      L)/50 mg/L=20 µL.

      **Step 3:** 20 µL of 50 mg/L
      kanamycin
      stock solution was added to each 20 mL LB liquid medium tube.
   5. **Preparation of LB Liquid Medium**

      **Objective:**

      Culture the BL21 strain containing pET-AsCpf1
      at 37
      ℃.

      **Step 1:** Inoculate each 20 mL
      LB
      culture tube with 1 mL of the overnight culture containing pET-AsCpf1 at a 5% inoculation rate (prepared in
      Experiment
      4, containing 50 mg/L kanamycin).

      **Step 2:** Incubate the culture
      at 37 ℃
      with shaking at 220 rpm to promote bacterial growth and amplification.
   6. **Monitor bacterial growth by measuring OD600 values.**

      This experiment aimed to monitor bacterial
      growth by
      regularly measuring the OD600 value of the culture using a microplate reader until it reached 0.6, indicating
      the log
      phase of growth. The procedure began with inoculation. 200 µL of bacterial suspension was transferred from the
      inoculation tube into a clear microplate well (LB medium) using a pipette. The mixture was thoroughly mixed
      and
      labeled as the experimental group (“CK”). The first OD600 measurement was taken immediately after inoculation.
      Subsequent measurements were taken at 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, and 3 hours after
      inoculation. Before each sampling, the culture was mixed to ensure uniformity. Once the OD600 value reached or
      exceeded 0.6, measurements were stopped, and the final data was recorded. Throughout the experiment, the
      culture was
      continuously shaken on a shaker, and all procedures were performed under aseptic conditions to avoid
      contamination.
   7. **Protein expression induced by different IPTG concentrations at 37 ℃**

      This experiment aimed to induce high-level
      protein
      expression at 37 ℃ using various IPTG concentrations.

      Six IPTG concentrations were used: 0 mM, 0.2
      mM, 0.5
      mM, 0.8 mM, 1 mM, and 2 mM, corresponding to sample IDs 37-1 through 37-6.

      The required volume of IPTG was calculated
      using the
      dilution formula: n=C1**×**V1=C2**×**V2.

      For example, for sample 37-2, which required
      a final
      IPTG concentration of 0.2 mM in 20 mL (V1 = 20 × 10³ µL, C1 = 0.2 mM), and with the IPTG stock concentration
      at 0.5 M
      (C2 = 0.5 × 10³ mM), the calculated volume (V2) was: V2 =
      (0.2**×**20**×**103)/(0.5**×**103) = 8 µL. Thus,
      8 µL of
      IPTG was added to the 20 mL culture.

      All samples were then incubated at 37 ℃ with
      shaking
      at 220 rpm for 3 hours.
   8. **Protein Expression Induced by Different IPTG Concentrations at 16 ℃**

      This experiment also aimed to induce
      high-level
      protein expression, but at a lower temperature (16 ℃).

      Six IPTG concentrations were used: 0 mM, 0.2
      mM, 0.5
      mM, 0.8 mM, 1 mM, and 2 mM, corresponding to sample IDs 16-1 through 16-6.

      As in Experiment 7, IPTG volumes were
      calculated
      using the formula: n=C1**×**V1=C2**×**V2. For instance, for sample 16-2, which
      required a
      final IPTG concentration of 0.2 mM in 20 mL, and a stock solution of 0.5 M: V2 =
      (0.2**×**20**×**103)/(0.5**×**103) = 8 µL.
      Therefore, 8
      µL of IPTG was added.

      The cultures were incubated at 16 ℃ with
      shaking at
      220 rpm for 20 hours.
2. **Results**
   1. **Monoclonal PCR validation results**

      ![](https://static.igem.wiki/teams/6004/notebook/7.webp)

      All the electrophoresis results showed bands
      corresponding to the expected size of the inserted fragments, indicating successful construction and
      transformation of
      the recombinant plasmid. The monoclonal PCR validation results demonstrated that the amplified AsCpf1 gene
      fragment
      was successfully inserted into the pET-28a vector. PCR analysis confirmed the presence of multiple positive
      clones,
      which were further verified through sequencing to ensure the accuracy of the recombinant plasmids. The results
      indicate that the target gene was successfully cloned and lays a foundation for subsequent protein expression.
      Meanwhile, the electrophoresis results also confirmed the successful construction of the A-Y, A-T, B-Y, and
      B-T
      gene-related recombinant plasmids, which are now available for further analysis.
   2. **Results of bacterial OD600 measurement**

      **Table 5. Bacterial
      OD600
      measurement results**

      |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      |  |  | **0 h** | **0.5 h** | **1 h** | **1.5 h** | **2 h** | **2.5 h** | **3 h** |
      | Group 37 ℃ | OD600 Value | 0.248 | 0. 254 | 0.293 | 0.364 | 0.472 | 0.606 | - |
      | Group 16 ℃ | OD600 Value | 0.189 | 0.238 | 0.276 | 0.336 | 0.445 | 0.550 | 0.586 |

      IPTG induction was performed after the
      OD600 value of the culture reached 0.6.

**Date: July, 2025**

1. **Experimental Contents**
   1. **Collect bacterial sedimentation**

      Centrifuge at 4 ℃, 5000 rpm for 15 min, and
      discard
      the upper culture medium.
   2. **Large-scale purification of His-tag protein (AsCpf1)**
   1. **Bacterial lysis**
   1. Add 5 mL of 1×PBS buffer to the bacterial sedimentation and fully resuspend the bacteria.
   2. Add 40 μL of lysozyme to a final concentration of 1 mg/ mL, mix well, and place on ice for 10 min.
   3. Sonicate the bacteria on ice. Set the sonication power to 50%, turn on for 3 s and turn off for 3 s, and
      process
      for 5 min.
   4. If the sonicated lysate is very viscous, repeatedly aspirate with a fine-needle syringe to shear the
      viscous
      genomic DNA. (optional)2. **Collect bacterial lysate**

      Centrifuge at 4 ℃, 5000 rpm for 10 min,
      collect the
      supernatant of the bacterial lysate, and place it on ice.
   3. **Equilibrate protein purification resin:**

      Take 1 mL of well-mixed 50% Beyogold
      His-tag
      Purification Resin, centrifuge at 4 ℃ (1000 ×g, 10 s), and discard the storage solution. Add 0.5 mL of
      non-denaturing
      lysis buffer to equilibrate the gel resin, centrifuge, discard the liquid, and equilibrate 1-2 more times,
      discarding
      the liquid each time.
   4. **Purify the protein**
   1. Mix 5 mL (all) of the bacterial lysate supernatant with the equilibrated Resin, and shake slowly at 4 ℃
      for 60
      min.
   2. Load the mixture of bacterial lysate and Resin into an empty affinity chromatography column.
   3. Open the cap at the bottom of the purification column, allow the liquid to flow out under gravity, and
      collect
      about 20 µL of the flow-through for analysis.
   4. Wash the column 5 times, adding 0.5-1 mL of non-denaturing washing buffer each time.
   5. Elute the target protein 6-10 times, using 0.5 mL of non-denaturing elution buffer each time.3. **Preparation of SDS-PAGE 10% protein gel (1.00 mm thick) using SDS-PAGE Color Preparation kit**
      1. Assemble the gel-making mold properly.
      2. Make a lower-layer gel by adding 2.7 mL of lower pre-mixed buffer reagents and SDS, and 55 μL improved
         coagulant
         into a glue mixing cup, stirring gently until evenly mixed and not generating bubbles.
      3. Add the lower-layer gel solution to the gel-making mold and keep the liquid level 1.5 cm away from the
         upper edge
         of the glass plate. Cover the solution with a layer of ethanol to keep the gel surface smooth and free of
         bubbles.
      4. Place it at room temperature for 8 minutes and wait for the gel to solidify.
      5. Make the upper layer gel by adding 1.5 mL lower pre-mixed buffer reagents and SDS, and 40 μL improved
         coagulant
         into a new glue mixing cup, stirring gently until evenly mixed and not generating bubbles.
      6. Add the upper layer gel solution to the gel-making mold above the lower layer gel until the gel solution
         reaches
         the top of the glass plate, then slowly insert the 1.00 mm thick comb into the gel and avoid air bubbles.
      7. Leave it for 15 minutes and wait for the upper gel to solidify. Gently remove the comb. Use a pipette or
         syringe
         to aspirate the electrophoresis buffer and rinse the sample wells clean.
   4. **Prepare the protein loading solution**

      Extract 16 μL purified protein under every
      concentration into a PCR tube alternatively, and add 4 μL 5**×** loading buffer into each tube.
      Boil at
      100 ℃ for 10 minutes. After centrifugation, the supernatant is used for sample loading (Do not suck up the
      sediment).

      The different concentrations include:

      37 ℃: 0 mM, 0.2 mM, 0.5 mM, 0.8 mM, 1 mM, 2
      mM

      16 ℃: 0 mM, 0.2 mM, 0.5 mM, 0.8 mM, 1 mM, 2
      mM
   5. **Prepare Tris-Glycine-SDS Buffer**

      Prepare Tris-Glycine-SDS Buffer used for
      SDS-PAGE
      electrophoresis by adding a packet of Tris-Glycine-SDS Buffer powder into 1 L ddH2O and mixing them
      evenly.
   6. **SDS-PAGE Gel Electrophoresis**
      1. Remove the SDS-PAGE gel from the gel casting plate.
      2. Place it into the inner chamber of the electrophoresis tank, with two gels facing each other and the
         shorter
         plates inward.
      3. Add SDS-PAGE electrophoresis buffer to the inner chamber.
      4. Slowly and vertically pull out the comb upward with both hands.
      5. Load samples into each well of the gel in the following order: Marker, 0 mM, 0.2 mM, 0.5 mM, 0.8 mM, 1 mM,
         and 2
         mM. The used marker band range was 10–180 kDa, and the predicted size of the target protein is 151.65 kDa.
      6. Run the gel at 180 V for 40 minutes in the electrophoresis tank.
   7. **SDS-PAGE Gel Staining**
      1. Use a gel spatula to separate the gel from the sides of the casting plate, then carefully lift and remove
         the
         post-electrophoresis SDS-PAGE gel.
      2. Stain the gel overnight with Coomassie Brilliant Blue R-250 fast staining solution (due to the staining
         solution
         being too dilute) under gentle shaking.
   8. **Destaining (Do it the next day.)**

      Destain the gel slowly with pure water on a
      horizontal shaker.
   9. **Observation of SDS-PAGE gel (Do it the next day.)**

      Photograph the stained and destained SDS-PAGE
      gel on
      a white background and save the images.
2. **Results**

   SDS-PAGE gel was stained overnight, and the
   results
   were observed the next day.

**Date: July, 2025**

1. **Experimental Contents**
   1. **Preparation of SDS-PAGE gels and SDS-PAGE gel electrophoresis**

      The gel was damaged, so SDS-PAGE gel
      electrophoresis
      was performed again with purified protein.

      For detailed operating steps, refer to
      yesterday\'s
      instructions. The simplified operating steps are as follows:
   1. Preparation of SDS-PAGE gel.
   2. Preparation of protein loading solution.
   3. Preparation of Tris-glycine-SDS buffer.
   4. SDS-PAGE electrophoresis.
   5. Staining of SDS-PAGE gel.
   6. Decolorization of gel.
   7. Observe and save the results of the SDS-PAGE gel.2. **Conduct an RPA experiment.**

      The primers used for the RPA reaction are:
      RPA-1M-A-F/R, RPA-2M-A-F/R, RPA-3M-A-F/R, RPA-4M-A-F/R, RPA-1M-B-F/R, RPA-2M-B-F/R, RPA-3M-B-F/R, and
      RPA-4M-B-F/R.
      RPA primer information is listed in the table below.

      **Table 6. RPA Primer
      information**

      |  |  |  |  |
      | --- | --- | --- | --- |
      | **Gene** | **Primer Name** | **Sequence (5’-3’)** | **Amplicon size** |
      | c .520G and c .520G>A | RPA-1M-c.520G>A-F | GACCTGGTGTCCCCAAGGTCTGCATCCTGA | 215 bp |
      | RPA-1M-c.520G>A-R | CAGTAGTGTCCTCAAGATGCTGAAGTCATTGA |
      | RPA-2M-c.520G>A-F | GAAGGGGCAGGGGGTCAAGCTATTTGCTGTC | 148 bp |
      | RPA-2M-c.520G>A-R | GGAAACGAGGGGCAGTAGTGTCCTCAAGATG |
      | RPA-3M-c.520G>A-F | GAAGGGGCAGGGGGTCAAGCTATTTGCTGAC | 148 bp |
      | RPA-3M-c.520G>A-R | GGAAACGAGGGGCAGTAGTGTCCTCAAGATG |
      | RPA-4M-c.520G>A-F | GAAGGGGCAGGGGGTCAAGCTATTTGCTCAC | 148 bp |
      | RPA-4M-c.520G>A-R | GGAAACGAGGGGCAGTAGTGTCCTCAAGATG |
      | c.6745C and c.6745C>T | RPA-1M-c.6745C>T-F | CTGACTGGACCTACTGGAGCTGTGGGACTTC | 164 bp |
      | RPA-1M-c.6745C>T-R | CTCCCTCTGTCTCCATCTTTTCCACTGGCAC |
      | RPA-2M-c.6745C>T-F | TTGTGGGTCCACAGGGGTCTCCAGGTTTGCCTGGC | 113 bp |
      | RPA-2M-c.6745C>T-R | AGGGCTCCCTCTGTCTCCATCTTTTCCACTG |
      | RPA-3M-c.6745C>T-F | TTGTGGGTCCACAGGGGTCTCCAGGTTTGCCTGCT | 113 bp |
      | RPA-3M-c.6745C>T-R | AGGGCTCCCTCTGTCTCCATCTTTTCCACTG |
      | RPA-4M-c.6745C>T-F | TTGTGGGTCCACAGGGGTCTCCAGGTTTGCCTCCT | 113 bp |
      | RPA-4M-c.6745C>T-R | AGGGCTCCCTCTGTCTCCATCTTTTCCACTG |

      The RPA reaction tubes contain white powder,
      which is
      the recombinant enzyme associated with the RPA reaction. The total volume of the RPA reaction is 50 μL,
      consisting of
      2.5 μL of upstream primer, 2.5 μL of downstream primer, 2 μL of activator (magnesium acetate), 20 μL of
      solvent, 21 μL
      of deionized water, and 2 μL of DNA template or deionized water (for the control group). It is important to
      note that
      2 μL of magnesium acetate should be added to the tube caps.

      For both A and B groups, each primer pair
      corresponds
      to three amplification reactions, with templates being plasmids containing normal sequences, plasmids with
      mutated
      sequences, and a blank control, respectively. After adding all components into the RPA reaction tubes
      containing the
      powder, close the tube caps and mix thoroughly. After briefly centrifuging, the mixture was incubated at 42 ℃
      for 20
      minutes.
   3. **Preparation of 3% agarose gel**

      Prepare the mesoporous agarose gel according
      to the
      following formula: 1.5 grams of agarose, 50 milliliters of TAE buffer, and 5 microliters of staining solution.
      Add the
      TAE buffer and agarose to a conical flask and mix thoroughly. Heat the mixture in a microwave until the
      solution
      becomes clear and boils. Then, add the staining solution and mix well.

      Pour the mixture into a medium-sized gel
      casting tray
      and insert a medium-sized comb. Allow the gel to cool before use.
   4. **Deactivating enzymes in RPA products**

      Inactivate the enzyme in RPA products
      according to
      the formula of 10 µL RPA products and 2 µL loading buffer, heat it at 65 ℃ for 5 min to denature the enzyme,
      and keep
      it at 12 ℃ until used.

      RPA product agarose gel electrophoresis
   5. **RPA product agarose gel electrophoresis**

      Load the processed RPA product samples into
      the wells
      of the gel, add DNA markers to the first and last wells of the gel, and run electrophoresis at 80 V for 1
      hour.
   6. **Observation of electrophoresis results using a gel imaging system**
2. **Results**
   1. **SDS-PAGE gel electrophoresis results for 16 ℃ induced expression**

      ![](https://static.igem.wiki/teams/6004/notebook/8.webp)

      The electrophoresis results of the crude
      protein
      demonstrate clear overexpression of the target protein (The left picture). Additionally, the intensity of
      protein
      bands in both the crude and purified protein samples follows a consistent trend with changes in IPTG
      concentration.
      After purification, the impurities present in the crude protein are nearly completely removed, leaving only
      the target
      protein.

      The results of induction at 16 ℃ with
      different IPTG
      concentrations are shown in the figure above. Upon examining the electrophoresis gel, it was observed that the
      overall
      band intensity was relatively high. As the IPTG concentration increased, the band intensity first increased
      and then
      stabilized. Under 16 ℃ cultivation conditions, the sample induced with 0.8 mM IPTG exhibited the strongest
      electrophoresis band, indicating that 0.8 mM IPTG is the optimal expression condition for cultivation at 16 ℃.
   2. **SDS-PAGE gel electrophoresis results for 37 ℃ induced expression**

      ![](https://static.igem.wiki/teams/6004/notebook/9.webp)

      The results of induction at 37 ℃ with different IPTG concentrations are shown in the figure above. Upon
      examining the
      electrophoresis gel, it was observed that the overall band intensity was weaker. As the IPTG concentration
      increased,
      the band intensity did not show significant changes. Under 37 ℃ cultivation conditions, the samples induced
      with 0.5
      mM and 2 mM IPTG showed relatively stronger bands, indicating that 0.5 mM and 2 mM IPTG are the optimal
      expression
      conditions for cultivation at 37 ℃.

      **Overall
      Conclusion**

      Based on the results, induction at 16 ℃ with
      0.8 mM
      IPTG was chosen as the optimal expression condition, as it provided the strongest expression bands, indicating
      a
      higher protein expression level.
   3. **RPA product electrophoresis results for c.520G and c.520G>A**

      ![](https://static.igem.wiki/teams/6004/notebook/10.webp)

      Note: A: c.520G>A

      In the electrophoresis image above, it can be
      observed that when using four different pairs of RPA primers, the target bands corresponding to the c.520G and
      c.520G>A templates are visible in the lanes of the RPA products, while the blank control group shows no
      bands. This
      indicates that both the normal and mutated sequences were successfully amplified, and there is no aerosol
      contamination. When using the RPA-1M-c.520G>A-F/R and RPA-2M-c.520G>A-F/R primers, the band intensity is
      brighter, whereas the bands corresponding to the RPA-3M-c.520G>A-F/R and RPA-4M-c.520G>A-F/R primers are
      relatively weaker. This is due to the latter primers introducing more mismatched sites, which can somewhat
      affect the
      RPA amplification efficiency
   4. **RPA product electrophoresis results for c.6745C and c.6745C>T**

      ![](https://static.igem.wiki/teams/6004/notebook/11.webp)

      Note: B: c.6745C>T

      The RPA amplification results for the c.6745C
      and
      c.6745C>T templates are similar to those for the c.520G and c.6745C>T templates. As seen in the
      electrophoresis
      image above, when using four different pairs of RPA primers, the RPA products corresponding to the c.6745C and
      c.6745C>T templates show target bands, while no bands are observed in the blank control group. This
      indicates that
      both the normal and mutated sequences were successfully amplified, and there is no aerosol contamination. When
      using
      the RPA-1M-c.6745C>T-F/R and RPA-2M-c.6745C>T-F/R primers, the band intensity is brighter, whereas the
      bands
      corresponding to the RPA-3M-c.6745C>T-F/R and RPA-4M-c.6745C>T-F/R primers are relatively weaker. This
      is due to
      the latter primers introducing more mismatched sites, which can somewhat affect the RPA amplification
      efficiency.

**Date: July, 2025**

1. **Experimental Contents**
   1. **Screening of Effective RPA Primers by Cas12a cleavage reactions**

      **Objective:**

      To screen RPA primers that can distinguish
      between
      normal and mutant sequences based on the fluorescence signal from Cas12a cleavage products. The selected
      primers will
      be used to construct a detection platform.

      **Procedure:**
   1. **RPA Amplification**

      Using different RPA primers, RPA
      amplification was
      carried out with c.520G, c.520G>A, c.6745C, and c.6745C>T sequences as templates. The amplification
      results were
      then validated.

      For detailed procedures, refer to
      yesterday\'s
      experiment.
   2. **Cas12a Cleavage of RPA Products**

      For each primer group, the corresponding
      RPA products
      for the c.520G and c.520G>A templates, as well as the c.6745C and c.6745C>T templates, were subjected
      to Cas12a
      cleavage reactions (with a fluorescence signal reporting probe FAM-ssDNA-BHQ1).

      In the Cas12a cleavage reaction system, for
      the
      c.520G and c.520G>A templates, crRNA-c.520G>A was used; for the c.6745C and c.6745C>T templates,
      crRNA-c.6745C>T was used.

      The 20 µL Cas12a cleavage reaction system
      includes:

      * 2 µL 10x Reaction Buffer
      * 0.67 µL 10 µM Cas12a protein
      * 0.4 µL 10 µM crRNA
      * 2 µL 10 µM ssDNA reporter
      * 2 µL RPA product
      * 0.25 µL RNase Inhibitor
      * 12.68 µL DEPC-treated water

      Each reaction was performed in triplicate.

      The reaction mixture was incubated at 40 ℃.
      For
      reactions with crRNA-c.520G>A, the reaction conditions were 40 ℃ for 5 minutes, followed by 85 ℃
      inactivation for
      10 minutes. For reactions with crRNA-c.6745C>T, the reaction conditions were 40 ℃ for 60 minutes,
      followed by 85 ℃
      inactivation for 10 minutes. After cooling the reaction products to room temperature, the results were
      analyzed.
   3. **Fluorescence Detection**

      **Objective:**

      To observe the fluorescence results and
      screen RPA
      primers that can effectively distinguish between normal and mutant sequences.

      **Procedure:**

      1. Add 180 µL of ddH₂O to each Cas12a cleavage products.
      2. Transfer the entire 200 µL solution into the wells of a non-transparent 96-well enzyme reaction plate.
      3. Perform fluorescence measurement using a microplate reader.

      Excitation wavelength: 492 nm,

      Emission wavelength: 522 nm.2. **Optimization of Reaction Time**

      **Objective:**

      To optimize the Cas12a cleavage reaction time
      to
      enhance the performance of the detection platform.

      **Procedure:**

      Based on the results of the previous
      experiment, the
      selected primers, RPA-2M-c.520G>A-F/R and RPA-4M-c.6745C>T, will be used for the subsequent experiments.

      Use RPA primers to amplify normal sequences
      and
      mutant sequences, and set up blank control groups. For c.520G and c.520G>A, each RPA product is subjected
      to six
      replicate Cas12a cleavage reactions. Each group included normal sequences, mutant sequences, and blank
      controls. The
      same samples were subjected to different Cas12a cleavage reaction times, followed by an 85 ℃ enzyme
      inactivation step.
      The reaction times (at 42 ℃) were set as 0 min, 1 min, 2 min, 3 min, 4 min, and 5 min.

      For c.6745C and c.6745C>T, the real-time
      fluorescent reaction device was used to monitor the effect of enzyme cleavage time on detection performance.
      The
      reaction time was 60 min.

      This section of the investigation includes
      the
      following experiments:

      1. RPA amplification
      2. Protein degradation in RPA products
      3. Agarose gel electrophoresis of RPA products
      4. Cas12a cleavage of RPA products
      5. Fluorescent detection of diluted Cas12a cleavage products

      These experiments have been conducted before.
      For
      detailed steps, it can be referenced from previous experiments.
   3. **Feasibility analysis of the detection platforms**

      **Objective:**

      To investigate the feasibility of an
      optimized
      detection system for single-base mutations associated with dystrophic epidermolysis bullosa (DEB).

      **Brief description:**

      For the two mutation sites c.520G>A and
      c.6745C>T, RPA amplification, Cas12a cleavage reaction, fluorescence detection, and test strip analysis
      were
      performed on normal and mutant sequences under optimized conditions.

      **Procedure:**

      1. **Fluorescence detection system**
      1. RPA amplification
      2. Protein degradation in RPA products
      3. Agarose gel electrophoresis of RPA products
      4. Cas12a cleavage of RPA products
      5. Fluorescent detection of diluted Cas12a cleavage products

      These experiments have been conducted
      before. For
      detailed steps, it can be referenced from previous experiments.

      2. **Test strip detection system**
      1. RPA amplification
      2. Protein degradation in RPA products
      3. Agarose gel electrophoresis of RPA products
      4. Cas12a cleavage of RPA products

      For strip testing, FAM-ssDNA-Biotin was
      substituted
      for ssDNA in the Cas12a cleavage reaction, compared to the fluorescent detection method.

      5. Test strip analysis of Cas12a cleavage products

      5 μL of Cas12a cleavage products were
      placed on the
      sample pad of the test strip. The test strip was then placed in 70 μL of 1× PBS buffer (containing 0.01%
      Tween-20) for
      chromatography. The appearance of a T line was observed within 2 min to determine the result.

      These experiments have been conducted
      before. For
      detailed steps, it can be referenced from previous experiments.
   4. **Gradient dilution of plasmids**

      According to calculations, for
      pET-28a-c.520G>A
      and pET-28a-c.6745C>T, 10 ng of plasmid contains 10⁹ copies of the target sequence. First, obtain a plasmid
      sample
      containing 10⁹ copies/μL, then perform a 10-fold serial dilution to obtain 10⁹ copies/μL, 10⁸ copies/μL, 10⁷
      copies/μL, 10⁶ copies/μL, 10⁵ copies/μL, 10⁴ copies/μL, 10³ copies/μL, 10² copies/μL, 10¹ copies/μL, and 10⁰
      copies/μL
      of pET-28a-c.520G>A and pET-28a-c.6745C>T plasmid samples.
2. **Results**
   1. **Cas12a cleavage fluorescence results of RPA products corresponding to
      different RPA primers for c.520G>A**

      ![](https://static.igem.wiki/teams/6004/notebook/12.webp)

      Based on the results from real-time
      fluorescence
      monitoring and endpoint fluorescence detection, for the c.520G>A mutation site, using the
      RPA-1M-c.520G>A-F/R
      primers, both normal and mutant sequences exhibited strong fluorescence signals. Although the signal from the
      mutant
      sequence was stronger than that of the normal sequence, there was no significant difference between them,
      indicating
      that this primer pair does not meet the detection requirements. When using RPA-2M-c.520G>A-F/R, the normal
      sequence
      showed almost no fluorescence signal, while the mutant sequence exhibited a strong fluorescence signal,
      effectively
      distinguishing between the mutant and normal sequences. Therefore, this primer can be used for the detection
      of the
      c.520G>A mutation associated with DEB. However, no fluorescence signal was observed when using
      RPA-3M-c.520G>A-F/R and RPA-4M-c.520G>A-F/R, suggesting that the excessive mismatches introduced by
      these
      primers prevented the crRNA from recognizing the target DNA sequence, thus failing to activate Cas12a cleavage
      activity.

      In conclusion, RPA-2M-c.520G>A-F/R primers
      were
      selected for the detection of the c.520G>A mutation site.
   2. **Cas12a cleavage fluorescence results of RPA products corresponding to different RPA primers for
      c.6745C>T**

      ![](https://static.igem.wiki/teams/6004/notebook/13.webp)

      For the c.6745C>T mutation site, real-time
      fluorescence monitoring and endpoint fluorescence detection results showed that using the
      RPA-1M-c.6745C>T-F/R,
      RPA-2M-c.6745C>T-F/R, and RPA-3M-c.6745C>T-F/R primers, both normal and mutant sequences exhibited
      strong
      fluorescence signals. Although the signal from the mutant sequence was stronger than that of the normal
      sequence,
      there was no significant difference between them, meaning these primers do not meet the detection
      requirements.
      However, when using RPA-4M-c.6745C>T-F/R, the normal sequence showed almost no fluorescence signal, while
      the
      mutant sequence showed a strong fluorescence signal, successfully distinguishing between the mutant and normal
      sequences. This primer can thus be used for detecting the c.6745C>T mutation site associated with DEB.
      Notably,
      when using the RPA-4M-c.6745C>T-F/R primer, the fluorescence signal from the mutant sequence significantly
      decreased. This is because the mismatches introduced by the RPA primer reduced the efficiency of crRNA
      recognition of
      the target DNA sequence, leading to a substantial decrease in Cas12a cleavage efficiency.

      In conclusion, RPA-4M-c.6745C>T-F/R
      primers were
      selected for the detection of the c.6745C>T mutation site.
   3. **Optimization of Cas12a cleavage reaction time for c.520G and c.520G>A**

      ![](https://static.igem.wiki/teams/6004/notebook/14.webp)

      Based on the results from real-time
      fluorescence
      monitoring and endpoint fluorescence detection, for the c.520G>A mutation site, a Cas12a cleavage reaction
      time of
      1 minute was sufficient to effectively distinguish between the mutant and normal sequences (with the mutant
      signal
      being more than 3 times that of the normal sequence). As the reaction time increased, both the normal and
      mutant
      sequence signals increased, with the signal from the mutant sequence showing a notably more significant
      enhancement,
      while the normal sequence signal increased more slowly. Between reaction times of 1 to 5 minutes, the normal
      and
      mutant sequences could be distinguished, indicating effective detection. Notably, at 3 minutes, the
      fluorescence
      signal difference between the normal and mutant sequences was the most pronounced. Given that the sample
      concentration
      of the normal sequence was 109 copies/μL, a relatively high sample level, the lack of strong
      fluorescence
      signals from the normal sequence was due to the difficulty of matching the RPA-amplified normal sequence with
      the
      crRNA, rather than low sample concentration.

      Therefore, 3 minutes was determined to be the
      optimal
      Cas12a cleavage reaction time for detecting the c.520G>A mutation site.
   4. **Optimization of Cas12a cleavage reaction time for c.6745C and c.6745C>T**

      ![](https://static.igem.wiki/teams/6004/notebook/15.webp)

      Based on the results from real-time
      fluorescence
      monitoring, for the c.6745C>T mutation site, the efficiency of Cas12a cleavage of RPA-amplified products
      was
      relatively low, which is consistent with the results observed during RPA primer screening. This is due to the
      introduction of additional mismatched sites by the RPA primers, which reduced the recognition efficiency of
      crRNA and
      the cleavage efficiency of Cas12a. As the reaction time increased, the signal from the mutant sequence
      continuously
      increased, reaching its maximum at 58 minutes, while the signal from the normal sequence remained unchanged
      over time.

      Therefore, 60 minutes was determined to be
      the
      optimal Cas12a cleavage reaction time for detecting the c.6745C>T mutation site.

      **Summary:**

      For the c.520G>A and c.6745C>T mutation
      sites,
      the RPA primers selected for the c.6745C>T mutation introduced more mismatched bases compared to the
      primers for
      the c.520G>A mutation, resulting in a greater impact on crRNA recognition and Cas12a cleavage efficiency.
      As a
      result, the fluorescence signal for c.6745C>T was relatively lower. However, based on the current results,
      the
      detection of the c.6745C>T mutation site can still be successfully carried out.
   5. **Feasibility analysis (fluorescence detection)**

      ![](https://static.igem.wiki/teams/6004/notebook/16.webp)

      As shown in the figure, for the c.520G>A
      and
      c.6745C>T mutation sites, the fluorescence signals from the mutant sequences were significantly higher than
      those
      from the normal sequences, indicating that the optimized fluorescence detection system can effectively
      distinguish
      between normal and mutant sequences. This detection system is feasible.
   6. **Feasibility analysis (test strip detection)**

      ![](https://static.igem.wiki/teams/6004/notebook/17.webp)

      As shown in the figure, for the c.520G>A and c.6745C>T mutation sites, the test strips for the mutant
      sequences
      display both a control line (C line) and a distinct test line (T line), while the normal sequences and blank
      controls
      only show the control line (C line). This indicates that the optimized test strip detection system can
      effectively
      distinguish between normal and mutant sequences, demonstrating the feasibility of this detection system.

'
},{
"title": "Date: August, 2025",
"content": '

1. **Experimental Contents**
   1. **Molecular sensitivity analysis**

      **Objective:**

      Investigate the sensitivity of the optimized
      detection system for analyzing single-nucleotide mutations associated with dystrophic epidermolysis bullosa
      (DEB).

      **Brief description:**

      For the two mutation sites c.520G>A and
      c.6745C>T, to investigate the analytical sensitivity of the detection system, plasmid samples containing
      different
      copy numbers of the mutated sequences were subjected to RPA amplification, Cas12a cleavage reaction,
      fluorescence
      detection, and test strip analysis under optimized conditions.

      The plasmid concentrations used were: 10⁸
      copies/μL,
      10⁵ copies/μL, 10⁴ copies/μL, 10³ copies/μL, 10² copies/μL, 10¹ copies/μL, and 10⁰ copies/μL.

      **Procedure:**

      1. **Fluorescence detection system**
      1. RPA amplification
      2. Protein degradation in RPA products
      3. Agarose gel electrophoresis of RPA products
      4. Cas12a cleavage of RPA products
      5. Fluorescent detection of diluted Cas12a cleavage products

      These experiments have been conducted
      before. For
      detailed steps, it can be referenced from previous experiments.

      2. **Test strip detection system**
         1. RPA amplification
         2. Protein degradation in RPA products
         3. Agarose gel electrophoresis of RPA products
         4. Cas12a cleavage of RPA products

         For strip testing, FAM-ssDNA-Biotin was
         substituted
         for ssDNA in the Cas12a cleavage reaction, compared to the fluorescent detection method.

         5. Test strip analysis of Cas12a cleavage products

         5 μL of Cas12a cleavage products were
         placed
         on the
         sample pad of the test strip. The test strip was then placed in 70 μL of 1× PBS buffer (containing 0.01%
         Tween-20) for
         chromatography. The appearance of a T line was observed within 2 min to determine the result.

         These experiments have been conducted
         before.
         For
         detailed steps, it can be referenced from previous experiments.
   2. **Random Sample Analysis**

      **Objective:**

      To investigate the actual sample analysis
      capability
      of the optimized detection system for detecting mutation sites associated with dystrophic epidermolysis
      bullosa (DEB).

      **Brief description:**

      For the two target sites, c.520G>A and
      c.6745C>T, standard plasmids containing both mutated and non-mutated sequences at varying concentrations
      (≥104 copies/µL) were randomly mixed. A total of 12 random samples were prepared, and the
      constructed
      method was used for analysis. All random samples were subjected to RPA amplification, Cas12a cleavage
      reaction,
      fluorescence detection, and test strip analysis under optimized conditions. Positive and negative controls
      were also
      set up in the experiment. PCR amplification is used to verify the presence of relevant genes (it cannot
      distinguish
      whether they are mutant sequences).

      **Procedure:**

      1. **Fluorescence detection system**
         1. RPA amplification
         2. Protein degradation in RPA products
         3. Agarose gel electrophoresis of RPA products
         4. Cas12a cleavage of RPA products
         5. Fluorescent detection of diluted Cas12a cleavage products

         These experiments have been conducted
         before. For
         detailed steps, it can be referenced from previous experiments.
      2. **Test strip detection system**
         1. RPA amplification
         2. Protein degradation in RPA products
         3. Agarose gel electrophoresis of RPA products
         4. Cas12a cleavage of RPA products

         For strip testing, FAM-ssDNA-Biotin was
         substituted
         for ssDNA in the Cas12a cleavage reaction, compared to the fluorescent detection method.

         5. Test strip analysis of Cas12a cleavage products

         5 μL of Cas12a cleavage products were
         placed on the
         sample pad of the test strip. The test strip was then placed in 70 μL of 1× PBS buffer (containing 0.01%
         Tween-20) for
         chromatography. The appearance of a T line was observed within 2 min to determine the result.

         These experiments have been conducted
         before. For
         detailed steps, it can be referenced from previous experiments.
2. **Results**
   1. **RPA product electrophoresis results for molecular sensitivity analysis for c.520G>A and
      c.6745C>T**
      ![](https://static.igem.wiki/teams/6004/notebook/18.webp)

      In the electrophoresis gel, the RPA
      amplification
      products of samples 108, 106, and 105 copies/µL showed bands that were
      consistent
      with the expected size. As the plasmid concentration decreased, the electrophoretic bands of the RPA products
      gradually weakened until they disappeared. This is due to the limited analytical capacity of RPA amplification
      combined with agarose gel electrophoresis. In summary, the appearance of the target band in the
      high-concentration
      samples and the absence of bands in the blank control group indicate that the RPA amplification reaction
      proceeded
      normally.
   2. **Molecular sensitivity analysis for c.520G>A (fluorescence detection)**

      ![](https://static.igem.wiki/teams/6004/notebook/19.webp)

      Analysis of the fluorescence detection
      results for
      the c.520G>A site revealed that as the plasmid concentration decreased, the fluorescence signal showed an
      overall
      decreasing trend. The fluorescence value of the 103 copies/µL sample showed a significant
      difference
      compared to the negative control, whereas the 102 copies/µL sample did not. This indicates that the
      detection limit of the fluorescence detection system for the c.520G>A site is 103 copies/µL.
   3. **Molecular sensitivity analysis for c.520G>A (test strip detection)**

      ![](https://static.igem.wiki/teams/6004/notebook/20.webp)

      Observing the test strip results for the
      c.520G>A
      site, it was found that as the plasmid concentration decreased, the strength of the T line on the test strip
      showed an
      overall decreasing trend. The 103 copies/µL sample displayed a clear T line, while the
      102
      copies/µL sample did not. This indicates that the detection limit of the test strip system for the c.520G>A
      site is
      103 copies/µL.
   4. **Molecular sensitivity analysis for c.6745C>T (fluorescence detection)**

      ![](https://static.igem.wiki/teams/6004/notebook/21.webp)

      Analysis of the fluorescence detection
      results for
      the c.6745C>T site showed that as the plasmid concentration decreased, the fluorescence signal overall
      exhibited a
      decreasing trend. The fluorescence value of the 105 copies/µL sample showed a significant
      difference from
      the negative control, while the 104 copies/µL sample displayed some fluorescence, but the data
      analysis
      revealed no significant difference compared to the negative control. This suggests that the detection limit of
      the
      fluorescence detection system for the c.6745C>T site is 105 copies/µL.
   5. **Molecular sensitivity analysis for c.6745C>T (test strip detection)**

      ![](https://static.igem.wiki/teams/6004/notebook/22.webp)

      Observing the test strip results for the
      c.6745C>T
      site, it was found that as the plasmid concentration decreased, the strength of the T line on the test strip
      also
      showed a decreasing trend. The 104 copies/µL sample displayed a clear T line, while the
      103
      copies/µL sample did not. This indicates that the detection limit of the test strip system for the
      c.6745C>T site
      is 104 copies/µL.
   6. **Random sample analysis for c.520G>A and c.6745C>T (fluorescence detection and test strip
      detection)**

      **![](https://static.igem.wiki/teams/6004/notebook/23.webp)**

      The results of the random sample analysis for
      the
      c.520G>A and c.6745C>T targets are displayed in the figure. Both the fluorescence detection and test
      strip
      detection methods yielded consistent results, effectively distinguishing the presence or absence of mutated
      sequences.
      This indicates that the detection system has the capability to analyze actual samples.
   7. **PCR validation of random samples**

      Based on the comparison of PCR results with
      fluorescence and test strip detection results, the following conclusions can be drawn:

      1. **COL7A1 c.520G>A**

      Samples 2, 3, 5, 7, 8, 10, 11, and 12
      contain the
      COL7A1 c.520 near gene segment. Among these, samples 2, 3, 7, 10, and 11 contain the COL7A1 c.520G>A
      (mutated
      sequence), while samples 5, 8, and 12 contain the COL7A1 c.520G (non-mutated sequence). Samples 1, 4, 6, and
      9 do not
      contain the COL7A1 c.520 near gene segment.

      ![](https://static.igem.wiki/teams/6004/notebook/24.webp)

      2. **COL7A1 c.6745C>T**

      Samples 2, 3, 5, 7, 10, 11, and 12 contain
      the COL7A1
      c.6745C near gene segment. Among these, samples 2, 3, 7, 10, and 11 contain the COL7A1 c.6745C>T (mutated
      sequence), while samples 5 and 12 contain the COL7A1 c.6745C (non-mutated sequence). Samples 1, 4, 6, 8, and
      9 do not
      contain the COL7A1 c.6745C near gene segment.

      ![](https://static.igem.wiki/teams/6004/notebook/25.webp)

      These results confirm that the detection
      system can
      accurately identify both the presence of the targeted gene segments and the mutations associated with
      COL7A1.

'
},{
"title": "Date: September, 2025",
"content": '

1. **Organize data and summarize experiments**

'
}]%}
{% block title %}notebook{% endblock %}
{% block lead %}
notebook
{% endblock %}

{% extends "layout.html" %}
{% set table\_data = [{
"title": "Overview",
"content": '

Dystrophic Epidermolysis Bullosa (DEB) is a genetic
disorder caused by mutations in the COL7A1 gene, leading to fragile skin, blisters, and severe complications.
Traditional diagnostic methods for DEB are invasive and expensive, creating the need for an accessible, non-invasive
approach. This project combines synthetic biology with CRISPR-Cas12a technology to create a non-invasive prenatal
diagnostic platform for DEB. We designed and constructed a novel plasmid vector for the expression of the Cas12a
protein, which was successfully expressed and purified in E. coli through a streamlined synthetic biology workflow.
The purified Cas12a enzyme was used in conjunction with Recombinase Polymerase Amplification (RPA) to detect two
high-frequency mutations in the COL7A1 gene (c.520G>A and c.6745C>T), which are major causes of DEB. By
extracting cell-free fetal DNA (cfDNA) from maternal blood, we were able to amplify and analyze the target mutations.
Cas12a’s unique trans-cleavage activity enabled the detection of mutation-specific signals, providing high sensitivity
and specificity. This CRISPR-based platform offers a low-cost, rapid, and non-invasive solution for early detection of
DEB, with the potential for wide clinical application. Through synthetic biology, this project demonstrates how
engineered proteins, such as Cas12a, can be harnessed to develop accessible diagnostic tools that improve healthcare
outcomes for rare genetic diseases.

In this study, we successfully constructed a series
of recombinant plasmids, namely pET28a-AsCpf1, pET28a-c.520G>A, pET28a-c.520G, pET28a-c.6745C>T, and
pET28a-c.6745C. These five plasmids were transformed into DH5α competent cells for subsequent large-scale
amplification of the target fragments. Additionally, pET28a-AsCpf1 was further transformed into BL21 competent cells
for protein expression. The purified AsCpf1 (Cas12a) protein underwent enzyme activity testing and was used as a key
component in the construction of the detection platform. The plasmids pET28a-c.520G>A, pET28a-c.520G,
pET28a-c.6745C>T, and pET28a-c.6745C were used as critical standard reference samples containing either normal or
mutant sequences in the detection process. These five plasmids are essential for Cas12a protein expression and the
establishment of the DEB detection platform. Their construction process will be detailed in this document.

**Figure 1**, using pET28a-AsCpf1 as an
example, demonstrates the key steps and operational procedures in plasmid construction. **Figure 2**
shows the principle of operation of the detection platform.

***![](https://static.igem.wiki/teams/6004/engineering/1.webp)***

**Figure 1. The plasmid construction process of
pET-28a-AsCpf1**

**![2](https://static.igem.wiki/teams/6004/engineering/2.webp)**

**Figure 2. Principle of the detection
platforms**

**Note：**

**A:** Principle of the fluorescence
detection platform.

**B:** Principle of the test strip
detection platform.

**Outline**

**Part 1:** pET28a-AsCpf1, BBa\_250ZZSEH
(Design; Build; Test; Learn)

**Part 2:** pET28a-c.520G, BBa\_25XMA9IE
(Design; Build; Test; Learn)

**Part 3:** pET28a-c.520G>A,
BBa\_25A2GFYI (Design; Build; Test; Learn)

**Part 4:** pET28a-c.6745C, BBa\_25WJ6OVG
(Design; Build; Test; Learn)

**Part 5:** pET28a-c.6745C>T,
BBa\_25LN1XT0 (Design; Build; Test; Learn)

'
},{
"title": "Explanation of each part",
"content": '',
"children": [{
"title": "Part 1: pET28a-AsCpf1, BBa\_250ZZSEH",
"content": '

**Design**

The pET28a plasmid is from our lab\'s stock, while
AsCpf1 is from a biotech company. To construct the plasmid pET-28a-AsCpf1, PCR amplification of the AsCpf1 gene was
performed. The pET-28a vector was then subjected to double digestion using BamH I and EcoR I enzymes. The gene
fragment was ligated to the linearized vector using a homologous recombination method, followed by transformation into
BL21 competent cells. Single colonies were picked from the transformation plate and verified by monoclonal PCR. The
amplified products were further sequenced and analyzed by sequence alignment to confirm successful construction of the
recombinant plasmid.

To achieve efficient Cas12a protein expression, the
recombinant plasmid pET28a-AsCpf1 was transformed into BL21 competent cells. To preserve the plasmids, they were also
transformed into DH5α cells. The subsequent AsCpf1 (Cas12a) proteins were extracted, purified, and used for further
testing and the construction of the detection platform.

**Build**

![3](https://static.igem.wiki/teams/6004/engineering/3.webp)

**Figure 3. Results of pET-28a-AsCpf1 plasmid construction**

**Note:**

**A:** Agarose gel electrophoresis
results of PCR amplification of the AsCpf1 gene. M: 5K DNA Marker, N: negative control.

**B:** Double digestion results of
pET-28a using BamH I and EcoR I.

**C:** Culture plate for transforming
competent cells with recombinant plasmids.

**D:** Monoclonal PCR verification of
transformation results.

**E.** Sequence alignment results of
recombinant plasmid pET-28a-AsCpf1.

The construction process is illustrated in
**Figure 1**. The relevant results of pET-28a-AsCpf1 plasmid construction are shown in **Figure
3**. As depicted in **Figure 3A**, the PCR amplification of the AsCpf1 gene produced a single
band around 4000 bp, which aligns with the expected size of 3936 bp, confirming successful amplification of the AsCpf1
gene. The double digestion results of the pET-28a vector are presented in **Figure 3B**. Upon examination
of the digestion products, the presence of the expected band size and comparison with other lanes verified successful
digestion. Subsequently, the recombinant plasmid was transformed into BL21 competent cells, and the appearance of
numerous single colonies on the transformation plate indicated a high transformation efficiency (**Figure
3C**). Transformation efficiency was further verified by monoclonal PCR, as shown in **Figure
3D**, where the expected band size was observed, confirming the presence of positive clones. Finally,
sequence analysis (**Figure 3E**) revealed that the amplified fragment matched the predicted sequence,
thus confirming the successful construction of the pET-28a-AsCpf1 plasmid.

**Test**

1. **Protein Expression**

   The protein expression of AsCpf1 (Cas12a) was carried
   out in the pET-28a-AsCpf1-BL21 strain.

   1. **Optimization of IPTG-induced expression at 16 ℃**

To optimize the IPTG-induced expression of AsCpf1,
pET-28a-AsCpf1-BL21 acterial cultures were grown to an OD600 of 0.6, followed by IPTG induction at
concentrations of 0 mM, 0.2 mM, 0.5 mM, 0.8 mM, 1 mM, and 2 mM. The cultures were then incubated at 16 ℃ with shaking
for 20 hours. After induction, the protein was purified using a protein purification kit, and both crude and purified
protein samples were analyzed by SDS-PAGE.

![4](https://static.igem.wiki/teams/6004/engineering/4.webp)

**Figure 4. IPTG-Induced Expression at 16 ℃ with different IPTG concentrations**

The electrophoresis results (**Figures 4A and
4B**) showed clear overexpression of the target protein in the crude sample, with increasing IPTG
concentrations leading to stronger protein bands. After purification, the impurities in the crude protein were almost
entirely removed, leaving predominantly the target protein. The intensity of the bands in both the crude and purified
samples followed a consistent trend with the increasing IPTG concentration. The sample induced with 0.8 mM IPTG
exhibited the strongest band, both in the crude and purified forms, indicating that 0.8 mM IPTG is the optimal
concentration for protein expression at 16 ℃.

2. **Optimization of IPTG-induced expression at 37 ℃**

For higher-temperature expression, cultures were
induced with IPTG at concentrations of 0 mM, 0.2 mM, 0.5 mM, 0.8 mM, 1 mM, and 2 mM, after reaching an
OD600 of 0.6. The cultures were incubated at 37 ℃ with shaking for 3 hours. Afterward, protein was purified
using the same protocol as the 16 ℃ induction, and the samples were analyzed by SDS-PAGE.

![](https://static.igem.wiki/teams/6004/engineering/5.webp)

**Figure 5. IPTG-Induced Expression at 37 ℃ with different IPTG concentrations**

The electrophoresis gel for the 37 ℃ induction
revealed weaker overall band intensities compared to the 16 ℃ induction (**Figure 5**). As the IPTG
concentration increased, the band intensity did not show significant changes. However, the samples induced with 0.5 mM
and 2 mM IPTG showed relatively stronger bands, suggesting these concentrations are optimal for expression at 37 ℃.
Although purification reduced some of the impurities, the overall protein expression at 37 ℃ was obviously weaker than
at 16 ℃.

3. **Induction temperature optimization**

![6](https://static.igem.wiki/teams/6004/engineering/6.webp)

**Figure 6. IPTG-Induced expression at different temperatures**

Comparing the results of IPTG induction at 16 ℃
(**Figure 6A**) and 37 ℃ (**Figure 6B**), it was observed that protein expression was
significantly higher at the lower temperature (16 ℃), especially at 0.8 mM IPTG. The overall band intensity at 16 ℃
was relatively high, and the purification process removed most impurities, leaving a clear target protein band. In
contrast, protein expression at 37 ℃ was weaker, even with higher IPTG concentrations, indicating that the lower
temperature (16 ℃) provides better conditions for AsCpf1 expression.

- **Protein functional validation**

  The AsCpf1 (Cas12a) protein, obtained through
  synthetic biology methods, was purified and used in combination with RPA for the construction of detection platforms
  targeting COL1A1 gene mutations c.520G>A and c.6745C>T for the diagnosis of dermatosparaxis Ehlers-Danlos
  syndrome (DEB). The detection process involves fetal DNA acquisition, RPA amplification, and Cas12a cleavage. Using
  different ssDNA reporters, we constructed both a fluorescence detection platform and a test strip detection platform.

  In our experiments, RPA primers were first screened,
  followed by optimization of the reaction time for Cas12a cleavage of RPA products. Ultimately, we finalized the
  components of the reaction system and established the detection platforms. During this process, the Cas12a protein,
  obtained through synthetic biology methods, demonstrated excellent cleavage activity. Part of the experimental
  results, which validate the Cas12a activity and the effectiveness of the detection platform we developed, are shown
  below.

  1. **Feasibility analysis of the detection platforms**

     For the two mutation sites c.520G>A and
     c.6745C>T, RPA amplification, Cas12a cleavage reaction, fluorescence detection, and test strip analysis were
     performed on normal and mutant sequences under optimized conditions.

  **2.1.1 Feasibility analysis for fluorescence detection platform**

  ![7](https://static.igem.wiki/teams/6004/engineering/7.webp)

  **Figure 7. Feasibility analysis for fluorescence detection platform**

  As shown in **Figure 7**, for the
  c.520G>A and c.6745C>T mutation sites, the fluorescence signals from the mutant sequences were significantly
  higher than those from the normal sequences, indicating that the optimized fluorescence detection system can
  effectively distinguish between normal and mutant sequences. This detection system is feasible.

  **2.1.2 Feasibility analysis for test strip detection platform**

  ![](https://static.igem.wiki/teams/6004/engineering/8.webp)

  **Figure 8. Feasibility analysis for test strip detection platform**

  As shown in **Figure 8**, for the
  c.520G>A and c.6745C>T mutation sites, the test strips for the mutant sequences display both a control line (C
  line) and a distinct test line (T line), while the normal sequences and blank controls only show the control line (C
  line). This indicates that the optimized test strip detection system can effectively distinguish between normal and
  mutant sequences, demonstrating the feasibility of this detection system.

  **Conclusion:**

  Both the fluorescence and test strip detection
  platforms effectively distinguish between normal and mutant sequences at the c.520G>A and c.6745C>T mutation
  sites. The optimized systems demonstrated clear differentiation, confirming their feasibility for mutation detection.
- **Molecular sensitivity analysis**

For the two mutation sites c.520G>A and
c.6745C>T, to investigate the analytical sensitivity of the detection system, plasmid samples containing different
copy numbers of the mutated sequences were subjected to RPA amplification, Cas12a cleavage reaction, fluorescence
detection, and test strip analysis under optimized conditions.

The plasmid concentrations used were: 10⁸ copies/μL,
10⁵ copies/μL, 10⁴ copies/μL, 10³ copies/μL, 10² copies/μL, 10¹ copies/μL, and 10⁰ copies/μL.

1. **RPA product electrophoresis results for molecular sensitivity analysis for c.520G>A and
   c.6745C>T**

**![9](https://static.igem.wiki/teams/6004/engineering/9.webp)**

**Figure 9. RPA product electrophoresis results
for molecular sensitivity analysis for c.520G>A and c.6745C>T**

In the electrophoresis gel (**Figure
9**), the RPA amplification products of samples 108, 106, and 105 copies/µL
showed bands that were consistent with the expected size. As the plasmid concentration decreased, the electrophoretic
bands of the RPA products gradually weakened until they disappeared. This is due to the limited analytical capacity of
RPA amplification combined with agarose gel electrophoresis. In summary, the appearance of the target band in the
high-concentration samples and the absence of bands in the blank control group indicate that the RPA amplification
reaction proceeded normally.

2. **Sensitivity analysis results of the fluorescence detection platform for c.520G>A**

![](https://static.igem.wiki/teams/6004/engineering/10.webp)

**Figure 10. Sensitivity analysis results of the fluorescence detection platform for c.520G>A**

Analysis of the fluorescence detection results for
the c.520G>A site revealed that as the plasmid concentration decreased, the fluorescence signal showed an overall
decreasing trend. The fluorescence value of the 103 copies/µL sample showed a significant difference
compared to the negative control, whereas the 102 copies/µL sample did not (**Figure 10**).
This indicates that the detection limit of the fluorescence detection system for the c.520G>A site is
103 copies/µL.

3. **Sensitivity analysis results of the test strip detection platform for c.520G>A**

![](https://static.igem.wiki/teams/6004/engineering/11.webp)

**Figure 11. Sensitivity analysis results of the test strip detection platform for c.520G>A**

Observing the test strip results in **Figure
11** for the c.520G>A site, it was found that as the plasmid concentration decreased, the strength of the
T line on the test strip showed an overall decreasing trend. The 103 copies/µL sample displayed a clear T
line, while the 102 copies/µL sample did not. This indicates that the detection limit of the test strip
system for the c.520G>A site is 103 copies/µL.

4. **Sensitivity analysis results of the fluorescence detection platform for c.6745C>T**

![](https://static.igem.wiki/teams/6004/engineering/12.webp)

**Figure 12. Sensitivity analysis results of the fluorescence detection platform for c.6745C>T**

The fluorescence detection results for the
c.6745C>T site in **Figure 12** showed that as the plasmid concentration decreased, the fluorescence
signal overall exhibited a decreasing trend. The fluorescence value of the 105 copies/µL sample showed a
significant difference from the negative control, while the 104 copies/µL sample displayed some
fluorescence, but the data analysis revealed no significant difference compared to the negative control. This suggests
that the detection limit of the fluorescence detection system for the c.6745C>T site is 105 copies/µL.

5. **Sensitivity analysis results of the test strip detection platform for c.6745C>T**

![](https://static.igem.wiki/teams/6004/engineering/13.webp)

**Figure 13. Sensitivity analysis results of the test strip detection platform for c.6745C>T**

Observing the test strip results for the c.6745C>T
site, it was found that as the plasmid concentration decreased, the strength of the T line on the test strip also
showed a decreasing trend. As shown in **Figure 13,** the 104 copies/µL sample displayed a
clear T line, while the 103 copies/µL sample did not. This indicates that the detection limit of the test
strip system for the c.6745C>T site is 104 copies/µL.

**Conclusion:**

The molecular sensitivity analysis reveals that the
fluorescence detection system for the c.520G>A site has a detection limit of 10³ copies/µL, while the test strip
system has the same limit. For the c.6745C>T site, the fluorescence detection system has a limit of 10⁵ copies/µL,
and the test strip system has a limit of 10⁴ copies/µL.

- **Random Sample Analysis**

For the two target sites, c.520G>A and
c.6745C>T, standard plasmids containing both mutated and non-mutated sequences at varying concentrations
(≥104 copies/µL) were randomly mixed. A total of 12 random samples were prepared, and the constructed
method was used for analysis. All random samples were subjected to RPA amplification, Cas12a cleavage reaction,
fluorescence detection, and test strip analysis under optimized conditions. Positive and negative controls were also
set up in the experiment. PCR amplification is used to verify the presence of relevant genes (it cannot distinguish
whether they are mutant sequences).

1. **Random sample analysis for c.520G>A and c.6745C>T (fluorescence detection platform and test strip
   detection platform)**

**![](https://static.igem.wiki/teams/6004/engineering/14.webp)**

**Figure 14. Random sample analysis results for
c.520G>A and c.6745C>T**

The results of the random sample analysis for the
c.520G>A and c.6745C>T targets are displayed in **Figure 14**. Both the fluorescence detection and
test strip detection methods yielded consistent results, effectively distinguishing the presence or absence of mutated
sequences. This indicates that the detection system can analyze actual samples.

2. **PCR validation of random samples**

![15](https://static.igem.wiki/teams/6004/engineering/15.webp)

**Figure 15. PCR validation results of random samples for c.520G>A and c.6745C>T**

Based on the comparison of PCR results (shown in
**Figure 15**) with fluorescence and test strip detection results, the following conclusions can be
drawn:

1. **COL7A1 c.520G>A**

Samples 2, 3, 5, 7, 8, 10, 11, and 12 contain the
COL7A1 c.520 near gene segment. Among these, samples 2, 3, 7, 10, and 11 contain the COL7A1 c.520G>A (mutated
sequence), while samples 5, 8, and 12 contain the COL7A1 c.520G (non-mutated sequence). Samples 1, 4, 6, and 9 do not
contain the COL7A1 c.520 near gene segment.

2. **COL7A1 c.6745C>T**

Samples 2, 3, 5, 7, 10, 11, and 12 contain the COL7A1
c.6745C near gene segment. Among these, samples 2, 3, 7, 10, and 11 contain the COL7A1 c.6745C>T (mutated
sequence), while samples 5 and 12 contain the COL7A1 c.6745C (non-mutated sequence). Samples 1, 4, 6, 8, and 9 do not
contain the COL7A1 c.6745C near gene segment.

These results confirm that the detection system can
accurately identify both the presence of the targeted gene segments and the mutations associated with COL7A1.

**Learn**

**Situation 1:**

**Problem:** During electrophoresis, we
observed that two samples were connected.

**Analysis:** The connection between the
two samples is likely due to the well being punctured or scratched during the loading process, causing the samples to
merge.

**Solution:** Carefully check the
integrity of the wells before loading the samples. Use a finer pipette tip and ensure gentle and precise loading to
avoid puncturing the wells.

**Situation 2:**

**Problem:** The plasmid extraction
concentration was low.

**Analysis:** The low plasmid
concentration may be due to the low transformation efficiency of the TOP10 cells, which could result in fewer plasmid
copies per cell.

**Solution:** Switch to DH5α competent
cells, which generally have higher transformation efficiency and a higher plasmid copy number.

**Situation3:**

**Problem:** During the purification
process, the wrong reagent was added. The elution buffer was mistakenly used as the wash buffer, causing the sample to
be discarded as waste.

**Solution:** Double-check the reagents
before use, ensuring that each reagent is properly labeled and used according to the protocol. Repeat the purification
process after correcting the mistake.

**Situation 4:**

**Problem:** During the experiment, we
observed that after the inoculated bacteria were spread on the culture dish and cultured, the bacterial density was
low, and there were scattered scratches on the solid culture dish.

**Analysis:** The low bacterial density
may be due to the insufficient concentration of bacteria applied to the culture dish. The scratches likely resulted
from excessive force during the coating process.

**Solution:** Centrifuge and resuspend
the bacterial pellet before coating to remove a portion of the supernatant, thereby increasing the bacterial
concentration. When applying the bacteria, slow down and ensure gentle handling to avoid damage to the surface.

**Situation 5:**

**Problem:** The bacterial culture
growth was too slow.

**Analysis:** After confirming that the
antibiotic was correctly added, the slow growth may be due to a low inoculation volume, poor strain vitality, or the
selection of a weak colony.

**Solution:** Increase the inoculation
volume or use a more vigorous strain. We used bacterial cultures from other team members. In the future, we will pay
closer attention when selecting colonies to ensure higher viability.

**Situation 6:**

**Problem:** Both the marker and the
sample were loaded onto the same SDS-PAGE gel.

**Solution:** Reload the samples,
ensuring that the marker and the samples are placed in separate lanes for clear and accurate comparison.

**Situation 7:**

**Problem:** During SDS-PAGE
electrophoresis, the migration speed was very slow.

**Analysis:** Upon checking the inner
chamber, the buffer level was found to have dropped, which slowed down the electrophoresis process.

**Solution:** Replenish the
electrophoresis buffer to the appropriate level and continue the electrophoresis. Monitor the process closely, adding
more buffer as needed to maintain proper electrophoretic conditions.

**Situation 8:**

**Problem:** During lateral flow strip
analysis, some samples did not show a C line within the first 20 seconds, although a faint C line appeared later.

**Analysis:** This result is related to
the detection principle of Cas12a-based test strips, where strong positive samples contain a high amount of broken
ssDNA (forming the T line) and a small amount of intact ssDNA (forming the C line). At the beginning of the assay,
there is not enough intact ssDNA accumulated at the C line, which causes the initial absence of the C line.

**Solution:** Allow more time for the
sample to run and accumulate more intact ssDNA for the C line to develop. For very strong positive samples, note that
the C line may even disappear, but the T line should be strong. If the T line is weak and no C line is present, the
result is invalid.

'
},{
"title": "Part 2: pET28a-c.520G,
BBa\_25XMA9IE",
"content": '

**Design**

The pET28a plasmid is from our lab\'s stock, while
gene c.520G (normal sequence) is from a biotech company. To construct the plasmid pET-28a-c.520G, PCR amplification of
the c.520G gene fragment was performed. The pET-28a vector was then subjected to double digestion using BamH I and
EcoR I enzymes. The amplified gene fragment was ligated into the linearized vector using a homologous recombination
method, followed by transformation into DH5α competent cells. Single colonies were picked from the transformation
plate and verified by monoclonal PCR. The amplified products were then sequenced and analyzed by sequence alignment to
confirm successful construction of the recombinant plasmid.

To obtain a large number of clones, the recombinant
plasmid pET-28a-c.520G was transformed into DH5α competent cells, resulting in the acquisition and preservation of the
strain pET-28a-c.520G-DH5α.

The construction process is illustrated in
**Figure 16**.

***![](https://static.igem.wiki/teams/6004/engineering/16.webp)***

**Figure 16. Results of pET-28a-c.520G plasmid
construction**

**Note:**

**A:** Agarose gel electrophoresis
results of PCR amplification of the c.520G gene. M: 2 K DNA Marker, N: negative control.

**B:** Double digestion results of
pET-28a using BamH I and EcoR I.

**C:** Culture plate for transforming
competent cells with recombinant plasmids.

**D:** Monoclonal PCR verification of
transformation results.

**E:** Sequence alignment results of
recombinant plasmid pET-28a-c.520G.

The relevant results of pET-28a-c.520G plasmid
construction are shown in **Figure 16**. As depicted in **Figure 16A**, the PCR
amplification of the c.520G gene produced a single band around 362 bp, which aligns with the expected size of 362 bp,
confirming successful amplification of the c.520G gene. The double digestion results of the pET-28a vector are
presented in **Figure 16B**. Upon examination of the digestion products, the presence of the expected
band size and comparison with other lanes verified successful digestion. Subsequently, the recombinant plasmid was
transformed into DH5α competent cells, and the appearance of numerous single colonies on the transformation plate in
**Figure 16C** indicated a high transformation efficiency. Transformation efficiency was further verified
by monoclonal PCR, as shown in **Figure 16D**, where the expected band size was observed, confirming the
presence of positive clones. Finally, sequence analysis (**Figure 16E**) revealed that the amplified
fragment matched the predicted sequence, thus confirming the successful construction of the pET-28a-c.520G plasmid.

**Test**

**Plasmid function verification**

![](https://static.igem.wiki/teams/6004/engineering/17.webp)

**Figure 17. Electrophoresis results of RPA products using pET-28a-c.520G and pET-28a-c.520G>A as the
template**

RPA amplification was performed using the
pET-28a-c.520G plasmid, followed by the construction and performance evaluation of the subsequent detection platform.
For example, in the amplification electrophoresis image shown in **Figure 17,** the expected-sized
amplification products were observed. Combined with sequencing results, this indicates that the plasmid can be used as
a standard reference sample for the DEB detection platform.

**Learn**

**Situation 1**

**Problem**: After grouping with other
teams, it is difficult to distinguish our PCR products.

**Analysis**: This could be due to a
lack of proper labeling, causing sample confusion.

**Solution**: Ensure clear and visible
labeling of each sample before loading them into the gel.

**Situation 2**

**Problem**: I observed similar
electrophoresis results for our PCR product and another one.

**Analysis**: The two genes differ by
only one nucleotide, so their amplification products are of the same size.

**Solution**: Understand the difference
between these products and confirm they are indeed from different genes. Further analysis can help identify their
differences.

**Situation 3**

**Problem**: The target band in the RPA
electrophoresis gel is hard to separate.

**Analysis**: RPA products are typically
small in size, making it difficult to resolve them properly.

**Solution**: Use a higher concentration
agarose gel for electrophoresis to improve resolution and achieve clearer separation of the target band.

**Situation 4**

**Problem**: When using a higher
concentration agarose gel, the marker does not spread well.

**Analysis**: Higher concentration gels
create more resistance during electrophoresis, which affects the migration.

**Solution**: Lower the electrophoresis
voltage and increase the running time to achieve better separation.

'
},{
"title": "Part 3: pET28a-c.520G>A,
BBa\_25A2GFYI",
"content": '

**Design**

The pET28a plasmid is from our lab\'s stock, while
gene c.520G>A (mutation sequence) is from a biotech company. For the construction of the pET-28a-c.520 G>A
plasmid, PCR amplification of the c .520G>A gene fragment was carried out. The pET-28a vector was then subjected to
double digestion using BamH I and EcoR I enzymes to linearize it. The amplified c.520G>A fragment was ligated into
the digested vector using a homologous recombination method, followed by transformation into DH5α competent cells.
After plating, individual colonies were selected, and monoclonal PCR was performed for initial screening. The cloned
products were sequenced and analyzed through sequence alignment to confirm the successful construction of the
recombinant plasmid.

To obtain many clones, the recombinant plasmid
pET-28a-c.520G>A was transformed into DH5α competent cells, resulting in the acquisition and preservation of the
strain pET-28a-c.520G>A-DH5α.

The construction process is illustrated in
**Figure 18**.

***![](https://static.igem.wiki/teams/6004/engineering/18.webp)***

**Figure 18. The plasmid construction process
of pET-28a-c.520G>A**

***![](https://static.igem.wiki/teams/6004/engineering/19.webp)***

**Figure 19. Results of pET-28a-c.520G>A
plasmid construction**

**Note:**

**A:** Agarose gel electrophoresis
showing the PCR amplification of the c.520G>A gene. M: 2K DNA Marker, N: negative control.

**B:** Double digestion results of
pET-28a vector using BamH I and EcoR I.

**C:** Transformation plate showing DH5α
competent cells containing recombinant plasmids.

**D:** Monoclonal PCR results for
transformation verification.

**E:** Sequence alignment results
confirming the recombinant plasmid.

Construct the plasmid pET-28a-c.520G>Aaccording to **Figure 18**. The detailed results for the pET-28a-c.520G>A plasmid
construction are presented in **Figure 19**. As shown in **Figure 19A**, the PCR
amplification of the c.520G>A gene generated a single band around 362 bp, which corresponds with the expected
fragment size, confirming successful gene amplification. The results of the double digestion of the pET-28a vector are
shown in **Figure 19B**, where the digestion products reveal the presence of the expected fragment sizes,
confirming proper digestion. The transformation of the recombinant plasmid into DH5α cells resulted in the formation
of numerous single colonies, as illustrated in **Figure 19C**, indicating high transformation efficiency.
The transformation was further confirmed by monoclonal PCR, shown in **Figure 19D**, where the expected
band size was detected, confirming the presence of positive clones. Lastly, sequence analysis in **Figure
19E** confirmed that the sequence of the amplified c.520G>A fragment matched the expected sequence,
validating the successful construction of the pET-28a-c.520G>A plasmid.

**Test**

**Plasmid function verification**

RPA amplification was performed using the
pET-28a-c.520G>A plasmid, followed by the construction and performance evaluation of the subsequent detection
platform. For example, in the amplification electrophoresis image shown in **Figure 17,** the
expected-sized amplification products were observed. Combined with sequencing results, this indicates that the plasmid
can be used as a standard reference sample for the DEB detection platform.

**Learn**

**Situation 1**

**Problem:** During SDS-PAGE gel
preparation, the solution started to solidify before being added to the glass plates.

**Analysis:** Too much polymerizing
agent was added, causing the solution to solidify too quickly.

**Solution:** Reduce the amount of
polymerizing agent to allow more time for the gel to be prepared.

**Situation 2**

**Problem:** After adding the lower gel
layer in SDS-PAGE preparation, I noticed the liquid level decreased.

**Analysis:** The gel plates were not
properly sealed, leading to leakage.

**Solution:** Always check for leaks
before pouring the gel, ensuring that the plates are tightly sealed.

**Situation 3**

**Problem:** After removing the comb in
SDS-PAGE preparation, the wells stuck together like bat wings.

**Analysis:** The comb was not inserted
properly, and when removed, the solution had already begun to solidify, causing the wells to merge.

**Solution:** Insert the comb back
quickly after removal before the solution solidifies, or clean the comb and reinsert it to avoid such issues.

'
}, {
"title":"Part 4: pET28a-c.6745C,
BBa\_25WJ6OVG",
"content": '

**Design**

The pET28a plasmid is from our lab\'s stock, while
gene c.6745C (normal sequence) is from a biotech company. For the construction of the pET-28a-c.6745C plasmid, a PCR
amplification was carried out to obtain the c.6745C gene fragment. The amplified product, which had a size of 310 bp,
was inserted into a pET-28a vector that had been digested with BamH I and EcoR I enzymes. This was followed by
ligation using a homologous recombination method, and the recombinant plasmid was then introduced into DH5α competent
cells. After transformation, individual colonies were picked, and monoclonal PCR was used for verification. The
amplified PCR products were sequenced and compared to confirm the successful creation of the recombinant plasmid.

To obtain many clones, the recombinant plasmid
pET-28a-c.6745C was transformed into DH5α competent cells, resulting in the acquisition and preservation of the strain
pET-28a-c.6745C-DH5α.

The construction process is illustrated in
**Figure 20**.

**![](https://static.igem.wiki/teams/6004/engineering/20.webp)**

**Figure 20. The construction procedure of the
pET-28a-c.6745C plasmid**

***![](https://static.igem.wiki/teams/6004/engineering/21.webp)***

**Figure 21. Results of pET-28a-c.6745C plasmid
construction**

**Note**:

**A:** PCR amplification results of the
c.6745C gene. M: 2K DNA Marker, N: negative control.

**B:** Double digestion of pET-28a
vector with BamH I and EcoR I enzymes.

**C:** Colony formation on the plate
after transformation with recombinant plasmids.

**D:** Monoclonal PCR verification of
the transformation results.

**E:** Sequence alignment of the
recombinant plasmid pET-28a-c.6745C.

Construct the plasmid pET-28a-c.6745Caccording to **Figure 20**. The results for the pET-28a-c.6745C plasmid construction are shown
in **Figure 21**. In **Figure 21A**, PCR amplification of the c.6745C gene produced a single
band around 310 bp, which matched the expected size, confirming the successful amplification of the c.6745C gene.
**Figure 21B** shows the results of the double digestion of the pET-28a vector. The digestion products
revealed the expected band sizes, confirming the success of the digestion process. Following this, the recombinant
plasmid was transformed into DH5α competent cells, and **Figure 21C** shows the appearance of numerous
colonies on the plate, indicating efficient transformation. Monoclonal PCR verification in **Figure 21D**
confirmed the presence of positive clones, as the expected band size was observed. Finally, the sequencing results
shown in **Figure 21E** indicated that the amplified fragment perfectly aligned with the expected
sequence, validating the successful construction of the pET-28a-c.6745C plasmid.

**Test**

**Plasmid function verification**

![](https://static.igem.wiki/teams/6004/engineering/22.webp)

**Figure 22. Electrophoresis results of RPA products using pET-28a-c.6745C and pET28a-c.6745C>T as the
template**

RPA amplification was performed using the
pET-28a-c.6745C plasmid, followed by the construction and performance evaluation of the subsequent detection platform.
For example, in the amplification electrophoresis image shown in **Figure 22,** the expected-sized
amplification products were observed. Combined with sequencing results, this indicates that the plasmid can be used as
a standard reference sample for the DEB detection platform.

**Learn**

**Situation 1**

**Problem:** The staining result with
Coomassie Brilliant Blue fast stain was not clear.

**Analysis:** The staining solution had
been reused multiple times, which affected its effectiveness.

**Solution:** We conducted overnight
staining and consider using fresh staining solution to improve the staining quality.

**Situation 2**

**Problem:** RPA electrophoresis showed
target bands as well as additional non-specific bands.

**Analysis**: RPA products are often
longer and more prone to primer-dimer formation or mismatches. Since we cannot distinguish normal and mutated
sequences by RPA amplification alone, this is not problematic for our experiment.

**Solution:** After the RPA reaction, we
added the Cas12a cleavage step to specifically identify the target sequence. The non-specific bands will not affect
the accuracy of the detection.

**Situation 3**

**Problem:** During the test strip
experiment, the wrong ssDNA was used for Cas12a cleavage.

**Analysis:** The ssDNA used for
fluorescence detection and for the test strip detection are different. Mixing them up will lead to erroneous results.
In a fluorescence detection system, ssDNA must be labeled with fluorescent groups and quenching groups so that a
fluorescent signal can be generated in the system after it is cleaved. For test strip detection systems, the ssDNA
label must correspond one-to-one with the antibody on the test strip; otherwise, the test strip analysis cannot be
performed normally. After using the wrong ssDNA, we repeated the experiment.

**Solution:** Ensure that the correct
ssDNA is used for each detection system. After the mistake, redo the experiment with the proper ssDNA.

'
},{
"title": "Part 5: pET28a-c.6745C>T,
BBa\_25LN1XT0",
"content": '

**Design**

The pET28a plasmid is from our lab\'s stock, while
gene c.6745C>T (mutation sequence) is from a biotech company. For the creation of the pET-28a-c.6745C>T plasmid,
the PCR amplification was performed using the c.6745C>T gene fragment. The amplified product, which had a size of
310 bp, was then inserted into a pET-28a vector previously digested with BamH I and EcoR I enzymes. The ligation step
was completed using a homologous recombination method, and the recombinant plasmid was subsequently introduced into
DH5α competent cells. After transformation, individual colonies were selected and monoclonal PCR was conducted to
verify the presence of the desired insert. The PCR products were sequenced and aligned to ensure the successful
construction of the plasmid.

To obtain many clones, the recombinant plasmid
pET-28a-c.6745C>T was transformed into DH5α competent cells, resulting in the acquisition and preservation of the
strain pET-28a-c.6745C>T-DH5α.

The construction process is illustrated in
**Figure 23**.

**![](https://static.igem.wiki/teams/6004/engineering/23.webp)**

**Figure 23. The construction process of the
pET-28a-c.6745C>T plasmid**

***![](https://static.igem.wiki/teams/6004/engineering/24.webp)***

**Figure 24. Results of the
pET-28a-c.6745C>T plasmid construction**

**Note:**

**A:** PCR amplification of the
c.6745C>T gene. M: 2K DNA Marker, N: negative control.

**B:** Double digestion of pET-28a
vector with BamH I and EcoR I.

**C:** Colony formation on the
transformation plate after recombinant plasmid introduction.

**D:** Monoclonal PCR verification of
the transformation.

**E:** Sequence alignment of the
recombinant plasmid pET-28a-c.6745C>T.

The plasmid construction process is shown in
**Figure 23**, with the corresponding results for the pET-28a-c.6745C>T plasmid construction displayed
in **Figure 24**. As seen in **Figure 24A**, PCR amplification of the c.6745C>T gene
generated a distinct band at 310 bp, confirming the successful amplification of the gene. The results of the double
digestion of the pET-28a vector are presented in **Figure 24B**, where the expected band sizes were
observed, validating successful digestion. After the recombinant plasmid was transformed into DH5α competent cells,
**Figure 24C** demonstrates the appearance of several colonies on the transformation plate, confirming
effective transformation. The monoclonal PCR results shown in **Figure 24D** further supported the
presence of positive clones, as indicated by the expected band. Lastly, the sequence alignment results in
**Figure 24E** confirmed that the amplified fragment matched the anticipated sequence, confirming the
successful construction of the pET-28a-c.6745C>T plasmid.

**Test**

**Plasmid function verification**

RPA amplification was performed using the
pET-28a-c.6745C>T plasmid, followed by the construction and performance evaluation of the subsequent detection
platform. For example, in the amplification electrophoresis image shown in **Figure 22,** the
expected-sized amplification products were observed. Combined with sequencing results, this indicates that the plasmid
can be used as a standard reference sample for the DEB detection platform.

**Learn**

**Situation 11**

**Problem:** Some PCR products from
different RPA primers showed weaker electrophoresis bands.

**Analysis:** The later RPA primers
introduced more mismatched sites, which is beneficial for distinguishing normal and mutated sequences in the Cas12a
step. However, these mismatches reduce the amplification efficiency.

**Solution:** The high sensitivity of
the Cas12a system compensates for this reduction in amplification efficiency, so continue using the system for
detection.

**Situation 12**

**Problem:** The detection of the
c.520G>A mutation took less time than the c.6745C>T mutation.

**Analysis:** The RPA primers for the
c.6745C>T target introduced more mismatched sites, which reduced the efficiency of crRNA recognition and Cas12a
cleavage.

**Solution:** Increase the Cas12a
cleavage time for detecting the c.6745C>T mutation to improve detection accuracy.

'
}]
},{
"title": "References and sources",
"content": '

[1] Xu, J.-M., Wu, Z.-S., Zhao, K.-J., Xi, Z.-J.,
Wang, L.-Y., Cheng, F., Xue, Y.-P. and Zheng, Y.-G. IPTG-induced high protein expression for whole-cell biosynthesis
of L-phosphinothricin. *Biotechnol. J.*, 18, 9 (2023), 2300027.

[2] Tan, M., Liao, C., Liang, L., Yi, X., Zhou, Z.
and Wei, G. Recent advances in recombinase polymerase amplification: Principle, advantages, disadvantages and
applications. *Frontiers in Cellular and Infection Microbiology*, Volume 12 - 2022 (2022-November-28 2022).

[3] Li, S.-Y., Cheng, Q.-X., Wang, J.-M., Li, X.-Y.,
Zhang, Z.-L., Gao, S., Cao, R.-B., Zhao, G.-P. and Wang, J. CRISPR-Cas12a-assisted nucleic acid detection. *Cell
Discovery*, 4, 1 (2018/04/24 2018), 20.

[4] Wang, B., Wang, R., Wang, D., Wu, J., Li, J.,
Wang, J., Liu, H. and Wang, Y. Cas12aVDet: a CRISPR/Cas12a-based platform for rapid and visual nucleic acid detection.
*Analytical chemistry*, 91, 19 (2019), 12156-12161.

[5] Ling, C., Chang, Y., Wang, X., Cao, X., Tu, Q.,
Liu, B. and Huang, S. Two CRISPR/Cas12a-based methods for fast and accurate detection of single-base mutations.
*Anal. Chim. Acta*, 1247 (2023/03/22/ 2023), 340881.

'
}]%}
{% block title %}engineering{% endblock %}
{% block lead %}
engineering
{% endblock %}

{% extends "layout.html" %}
{% set table\_data = [{
"title": "Preparation of LB Liquid and Solid Media",
"content": '

**1 L formula:**

|  |  |
| --- | --- |
| **Component** | **Weight (g)** |
| Tryptone | 10 |
| Yeast extract | 5 |
| NaCl | 10 |

**Procedure:**

1. Prepare the liquid and solid medium according to the system above
2. Only solid medium needs to be supplemented with AGAR powder, 15–20 g/L.
3. 121 ℃, 15–20 minutes.
4. Add 100 μLof 50 mg/mL Kana solution to every 100 mL medium for solid media.

'
},{
"title": "Prepare of 50 mg/mL Kanamycin (Kana) Stock Solution",
"content": '

To prepare 10 mL of 50 mg/mL Kanamycin (Kana) Stock Solution:

1. Weigh 500 mg of kanamycin sulfate.
2. Add 10 mL of sterile distilled water and mix until fully dissolved.
3. Filter the solution through a 0.22 μm sterile filter to remove potential contaminants.
4. Dispense into sterile microcentrifuge tubes and store at -20 ℃.

'
}, {
"title": "Inoculation of bacterial culture",
"content": '

**Procedure:**

1. All operations are performed in a sterile area.
2. Inoculate the culture medium with the inoculum at an appropriate ratio: 1:100.
3. Incubate the culture tubes on a bacterial incubator at 37 ℃ for 12–16 hours, shaking at a speed of 180–220 rpm.

'
},{
"title": "PCR Amplification",
"content": '

The reaction system is as follows:

|  |  |
| --- | --- |
| **Component** | ×1（50 µL） |
| 2x Taq Master Mix | 20 |
| primer F | 2.5 |
| primer R | 2.5 |
| DNA template | 2 |
| ddH2O | 21 |

**Procedure:**

95 ℃ for 1 minute. Cycle 30-35 times for: 95 ℃ for 30
seconds, 56 ℃ for 30 seconds, 72 ℃ for 4 minutes. 72 ℃ for 5 minutes. Then 12 ℃ until the PCR products are taken out.

Adjust the extension time appropriately for different
sizes of amplification products.

For single-clone PCR, pick a single colony from the
culture plate as the amplification template instead of the DNA template.

'
}, {
"title": "Preparation of Agarose Gel",
"content": '

**Materials:**

1**×**TAE buffer, agarose powder.

**Equipment:**

Gel casting tray, gel comb, electronic balance,
graduated cylinder, Erlenmeyer flask, micropipette.

**Formula:**

|  |  |  |  |
| --- | --- | --- | --- |
|  | **Small gel** | **Medium gel** | **Large gel** |
| Agarose | 0.45g | 0.75g | 1.5g |
| TAE buffer | 30mL | 50mL | 100mL |
| dye | 2μl | 4μl | 8μl |

**Procedure:**

1. **Calculation:**

For this experiment, a small gel casting tray is
used, requiring 30 mL of TAE buffer and 0.45 g of agarose powder.

2. **Measurement:**

Weigh approximately 0.45 g of agarose powder using an
electronic balance. Measure 30 mL of TAE buffer using a graduated cylinder and micropipette.

3. **Mixing:**

Add the agarose powder and TAE buffer into an
appropriately sized Erlenmeyer flask. Gently swirl the flask until the agarose powder partially dissolves. Then, place
the flask in a heating device (microwave) and heat until all agarose particles are completely dissolved. After
heating, the solution should appear clear.

4. **Staining:**

Add 2 µL of nucleic acid stain to the solution and
gently swirl until the solution shows a light pink color.

5. **Gel Casting:**

Slowly pour the prepared solution (agarose solution
with nucleic acid stain) into the gel casting tray to avoid bubble formation, which can interfere with observation.
Let the solution sit undisturbed until it solidifies into a gel.

'
}, {
"title": "Agarose gel electrophoresis",
"content": '

1. Add 1 μLof 6× loading buffer to each 5 μLof PCR product and centrifuge to mix.
2. Remove the comb from the agarose gel and place the gel in the electrophoresis chamber.
3. Add an appropriate amount of electrophoresis buffer to the electrophoresis chamber.
4. Load the mixture into the wells of the gel and add DNA markers to the wells at both ends of the sample.
5. Turn on the electrophoresis instrument and set the electrophoresis parameters: 120 V, 20 minutes.
6. Observe the results under a blue light transilluminator or gel imaging system, such as the Gel Doc™ XR+.

'
},{
"title": "Plasmid extraction",
"content": '

**Step 1:** Scientists took 3 mL of the
fifteen-hour cultured bacterial liquid and added it to a centrifuge tube. Then, it is centrifuged at 10,000 rpm
(11,500×g) for 1 minute. The culture medium was then discarded as much as possible.

**Step 2:** Scientists then added 250
μLof Buffer P1that contains RNase A, to the centrifuge tube with the bacterial pellet, and mixed well with a vortex
oscillator. This process resuspended the bacteria.

**Step 3:** Then, 250 μLof Buffer P2 is
added to the centrifuge tube and is mixed with gentle inversion 8-10 times to lyse the bacteria fully.

**Step 4:** Then, 350 μLof Buffer P3 is
added to the centrifuge tube, and is mixed with gentle inversion 8-10 times to neutralize Buffer P2 thoroughly. At
this point, there should be white flocculent precipitation. The tube is then centrifuged at 12,000 rpm (13,400×g) for
10 minutes.

**Step 5:** Then, Scientists place the
FastPure DNA Mini Columns in a 2 mL collection tube. The supernatant from the previous step is then transferred to the
adsorption column with a pipette, and later centrifuged at 12,000 rpm (13,400×g) for 45 seconds. The waste liquid in
the collection tube is then discarded. This process is meant for column loading and plasmid binding.

**Step 6:** Scientists then added 500
μLof Buffer PW1 to the adsorption column, which was centrifuged at 12,000 rpm (13,400×g) for 45 seconds. The waste
liquid is then discarded. This process is to wash off the protein.

**Step 7:** 600 μLof Buffer PW2 diluted
with anhydrous ethanol is then added to the adsorption column. Centrifuged at 12,000 rpm (13,400×g) for 45 seconds.
The waste liquid is then discarded.

**Step 8:** The process above is
repeated twice, washing off the salt ions.

**Step 9:** The adsorption column is
then placed back in the collection tube and centrifuged at 12,000 rpm (13,400×g) for 1 minute to dry the adsorption
column, removing the rest of the washing solution in the adsorption column.

**Step 10:** The adsorption column is
then placed in a new sterile 1.5 mL centrifuge tube. Then, 100 μLof Elution Buffer is added to the center of the
membrane of the adsorption column. The column is then placed at room temperature for 2 minutes, and centrifuged at
12,000 rpm (13,400×g) for 1 minute to elute the DNA. This process washes off the protein.

**Step 11:** The adsorption column is
then discarded, and the DNA product is stored at -20℃ to prevent DNA degradation.

'
}, {
"title": "Linearize plasmid",
"content": '

The reaction system is as follows:

|  |  |
| --- | --- |
| **Component** | **×1 (50 μL)** |
| plasmid pET-28a | 1 μg |
| EcoR I | 1 |
| BamH I | 1 |
| CUM Smart Buffer | 5 |
| ddH₂O | up to 50 μL |

**Procedure:**

1. Complete the whole experiment on ice.
2. Incubate the reaction mixtures at 37 ℃ for 10 minutes to complete the digestion and linearize the plasmid.

'
}, {
"title": "Purification of PCR products and restriction enzyme digestion products",
"content": '

**Process:**

1. Transfer 50 μLenzyme-digested product and 100 μLPCR product into two different adsorption columns
   respectively, and then 12,000 rpm (13,800 ×g) centrifuge 40 seconds. DNA is adsorbed on the membrane of the
   adsorption column.
2. Discard the filtrate and place the adsorption column in the collection tube. Add 300 μLof Buffer GDP to the
   adsorption column. Let it stand for 1 minute, and then 12,000 rpm (13,800 ×g) centrifuge for 40 seconds.
3. Discard the filtrate and place the adsorption column in the collection tube. Add 700 μLBuffer GW (anhydrous
   ethanol has been added) to the adsorption column. Cover the lid, invert the collection tube 3 times to help
   thoroughly rinse off the salt adhering to the pipe wall, and then 12,000 rpm (13,800 ×g) centrifuge 40 seconds.
4. Repeat step 3 to make sure the salt is completely removed.
5. Discard the filtrate and place the adsorption column in the collection tube. 12,000 rpm (13,800 ×g) centrifuge
   for 2 minutes.
6. Place the adsorption column in 1.5 mL sterilized centrifuge tubes. Add 30 μLddH2O into the middle
   of the adsorption column, place it for 2 minutes, and then 12,000 rpm (13,800 ×g) centrifuge 1 minute. Discard
   the adsorption column, detect the concentration of the products, and store the DNA at -20℃.

'
},{
"title": "Homologous Recombination-based Ligation",
"content": '

The reaction system is as follows:

|  |  |
| --- | --- |
| **Component** | **×1 (10 μL)** |
| 2×Clon Express Mix | 5 μL |
| Linearized vector pET-28a | 0.03 pmol |
| Insert fragments | 0.06 pmol |
| ddH₂O | up to 10 μL |

**Procedure:**

1. Assemble the reaction system on ice.
2. Gently mix by pipetting up and down (avoid vortexing), then briefly centrifuge to collect the reaction mixture at
   the bottom of the tube.
3. Incubate at 50 ℃ for 15 minutes; then reduce the temperature to 4 ℃ or immediately place on ice for cooling.

'
}, {
"title": "Heat Shock Transformation",
"content": '

**Materials:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Recombinant product | 10 μL |
| Competent cell | 100 mL |
| LB culture medium | 890 μL |

**Procedure:**

1. Thaw competent cells on ice.
2. Add 10 μL recombinant product to 100 μL competent cells, mix gently by flicking the tube (do not vortex), and
   incubate on ice for 30 minutes.
3. Heat-shock at 42 ℃ water bath for 45 seconds.
4. Place immediately on ice for 1 minute.
5. Add 890 μLLB medium (without antibiotics) and incubate at 37 ℃ for 60 minutes.
6. Spread 100 μL bacterial culture via the spread plate method.
7. Incubate at 37 ℃ for 12–16 hours.

'
}, {
"title": "Microbial culture using the LB agar plate method",
"content": '

**Procedure:**

1. Prepare a solid culture medium and sterilize it.
2. Allow the culture medium to cool to 60 ℃, then pour it into sterile agar plates.
3. Once the medium in the plates has solidified, use a pipette to add 50–100 μL of the culture to the center of the
   plate.
4. Use a spreader to evenly spread the bacteria across the surface of the medium.
5. Invert the plates and incubate at 37 ℃ for 16–24 hours.
6. Observe and record the growth status of the bacteria.

'
},{
"title": "Prepare 40% glycerol",
"content": '

Prepare 200 mL of glycerin with a concentration of
40%: 80 mL glycerol plus 120 mL of ddH2O, then high-temperature and high-pressure sterilization (121 ℃, 20
min).

'
},{
"title": "Bacterial culture preservation",
"content": '

**Materials:** bacterial culture.

**Reagent:** 40% glycerol.

**Procedure:**

1. Add 500 μL of bacterial culture and 500 μL of 40% glycerol to a sterile centrifuge tube.
2. Mix the bacterial culture and glycerol thoroughly.
3. Store it at -80 ℃.

'
}, {
"title": "Bacterial Inoculation (from monoclonal to culture broth)",
"content": '

**Procedure:**

1. Using a sterile pipette tip, pick a single bacterial colony.
2. Dip the tip into a tube containing 3 mL LB medium and vigorously vortex or flick the tube to dislodge the
   bacteria.
3. Place the inoculated tube in a 37 ℃ shaking incubator for 12–16 hours.

'
}, {
"title": "Bacterial OD600 Measurement Protocol Using a Microplate Reader",
"content": '

**Materials:** bacterial
culture,
microplate reader, 96-well microplate, culture medium

**Protocol:**

1. **Prepare the microplate reader:**

Turn on the microplate reader and set it to measure
absorbance at 600 nm (OD600). Ensure that the instrument is properly calibrated.

2. **Blank the microplate reader:**

Add PBS or the same culture medium used for bacterial
growth into one well of the microplate (typically 200 µL). This will act as the blank.

3. **Prepare bacterial culture:**

Shake or vortex the bacterial culture gently to
ensure it is homogenous.

4. **Add bacterial culture to the microplate:**

Pipette an appropriate volume (typically 200 µL) of
bacterial culture into the wells of the microplate.

5. **Measure the OD600:**

Place the microplate into the microplate reader and
measure the absorbance at 600 nm (OD600). Record the values for each well.

6. **Check bacterial growth stage:**

The OD600 value correlates with bacterial cell
concentration. Typically:

OD600 ~0.1-0.3: Log phase (rapid growth)

OD600 ~0.5-1.0: Mid-log phase

OD600 >1.0: Stationary or early stationary phase

'
},{
"title": "IPTG-induced protein expression",
"content": '

**Procedure**

1. Expand the bacterial culture to an OD600 of 0.6.
2. Add IPTG to the bacterial culture to achieve the appropriate final concentration.

If investigating the optimal IPTG concentration, set
six different concentrations, such as 0 mM, 0.2 mM, 0.5 mM, 0.8 mM, 1 mM, and 2 mM, corresponding to different sample
numbers.

The required IPTG volume is calculated using the
dilution formula: n = c₁ × v₁ = c₂ × v₂.

For example, for sample 37-2, the final IPTG
concentration is 0.2 mM, with a volume of 20 mL (v₁ = 20 × 10³ μL, c₁ = 0.2 mM), and the IPTG stock solution
concentration is 0.5 M (c₂ = 0.5 × 10³ mM), the calculated volume (v₂) is: v₂ = (0.2 × 20 × 10³) / (0.5 × 10³) = 8 μL.
Therefore, add 8 μL of IPTG to 20 mL of LB medium.

1. Incubate all samples at 37 ℃ with shaking at 220 rpm for 3 hours, or at 16 ℃ with shaking at 220 rpm for 20 hours.

'
},{
"title": "Perform protein purification using the Beyotime Protein Purification Kit",
"content": '

**Materials:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Bacterial culture | 20 mL |
| non-denaturing lysis buffer | 500 μL |
| lysozyme | 1 mg/mL, 40 μL |
| BeyoGold™ His-tag Purification Resin (reduction-resistant chelating type) | 1 mL |
| non-denaturing wash buffer | 1 mL |
| non-denaturing elution buffer | 0.5 mL |

**Procedure:**

1. **Bacterial lysis**

1. Add 5 mL of 1×PBS buffer to the bacterial sedimentation and fully resuspend the bacteria.
2. Add 40 μL of lysozyme to a final concentration of 1 mg/ mL, mix well, and place on ice for 10 min.
3. Sonicate the bacteria on ice. Set the sonication power to 50%, turn on for 3 s and turn off for 3 s, and process
   for 5 min.
4. If the sonicated lysate is very viscous, repeatedly aspirate with a fine-needle syringe to shear the viscous
   genomic DNA. (optional)

2. **Collect bacterial lysate**

Centrifuge at 4 ℃, 5000 rpm for 10 min, collect the
supernatant of the bacterial lysate, and place it on ice.

3. **Equilibrate protein purification resin:**

Take 1 mL of well-mixed 50% Beyogold His-tag
Purification Resin, centrifuge at 4 ℃ (1000 ×g, 10 s), and discard the storage solution. Add 0.5 mL of non-denaturing
lysis buffer to equilibrate the gel resin, centrifuge, discard the liquid, and equilibrate 1-2 more times, discarding
the liquid each time.

4. **Purify the protein**

1. Mix 5 mL (all) of the bacterial lysate supernatant with the equilibrated Resin, and shake slowly at 4 ℃ for 60
   min.
2. Load the mixture of bacterial lysate and Resin into an empty affinity chromatography column.
3. Open the cap at the bottom of the purification column, allow the liquid to flow out under gravity, and collect
   about 20 µL of the flow-through for analysis.
4. Wash the column 5 times, adding 0.5-1 mL of non-denaturing washing buffer each time.
5. Elute the target protein 6-10 times, using 0.5 mL of non-denaturing elution buffer each time.

'
}, {
"title": "RPA Reaction",
"content": '

The reaction system is as follows:

|  |  |
| --- | --- |
| **Component** | **×1 (50 μL)** |
| dissolving agent | 20 |
| activator | 2 |
| Primer-F | 2.5 |
| Primer-R | 2.5 |
| DNA template | 2 |
| ddH₂O | 21 |

**Procedure:**

The RPA reaction tube contains white powder, which is
a recombinase associated with the RPA reaction. The proportions of each component are based on the reaction system.
Please note that 2 μL of magnesium acetate should be added to the tube cap.

After adding all components to the RPA reaction tube
containing the powder according to the reaction system, close the tube cap and mix thoroughly. After briefly
centrifuging, incubate the mixture at 42 ℃ for 20 minutes.

'
}, {
"title": "Cas12a cleavage reaction",
"content": '

The reaction system is as follows:

|  |  |
| --- | --- |
| **Component** | **×1 (20 μL)** |
| 10× Reaction buffer | 2 μL |
| Cas12a (10 μM) | 0.67 μL |
| crRNA (10 μM) | 0.4 μL |
| ssDNA (10 μM) | 2 μL |
| RPA product | 2 μL |
| RNase inhibitor | 0.25 μL |
| DEPC-treated water | 12.86 μL |

**Procedure:**

The reaction mixture is incubated at 40 ℃ for 5–60
minutes, followed by inactivation at 85 ℃ for 10 minutes. The reaction product is cooled to room temperature before
the results are analyzed.

For fluorescence detection systems, FAM-ssDNA-BHQ1
was used.

For strip testing, FAM-ssDNA-Biotin was substituted
for ssDNA in the Cas12a cleavage reaction, compared to the fluorescent detection method.

'
}, {
"title": "Fluorescence detection of the Cas12a cleavage product",
"content": '

**Procedure:**

1. Add 180 µL of ddH₂O to each Cas12a cleavage product.
2. Transfer the entire 200 µL solution into the wells of a non-transparent 96-well enzyme reaction plate.
3. Perform fluorescence measurement using a microplate reader.

Excitation wavelength: 492 nm,

Emission wavelength: 522 nm.

'
},{
"title": "Test strip analysis of Cas12a cleavage products",
"content": '

5 μL of Cas12a cleavage products were
placed on the
sample pad of the test strip. The test strip was then placed in 70 μL of 1× PBS buffer (containing 0.01% Tween-20) for
chromatography. The appearance of a T line was observed within 2 min to determine the result.

'
},{
"title": "Preparation of the standard sample to be tested",
"content": '

1. **Extraction of plasmid**

**Procedure:**

1. Pick single colonies from transformation plates and inoculate them in liquid culture.
2. Inoculate the gum tip into 3 mL liquid culture medium in a tube. Place it on a shaker for cultivation for 12–16
   hours (220 rpm).
3. Extract plasmid pET-28a-c .520G, pET-28a-c .520G>A, pET-28a-c.6745C, pET-28a-c.6745C>T from their bacteria
   solution.

2. **Dilution of plasmid**

**Procedure:**

Dilute the plasmid to obtain gradient samples with
concentrations of
108、107、106、105、104、103、102、101、100
copies/Μl.

3. **Preparation of random samples**

**Procedure:**

For the two target sites, c.520G>A and
c.6745C>T, standard plasmids containing both mutated and non-mutated sequences at varying concentrations (≥104
copies/µL) were randomly mixed. A total of 12 random samples were prepared.

'
},{
"title": "Procedure of the detection platforms",
"content": '

**Procedure:**

1. **Fluorescence detection system**

1. RPA amplification
2. Protein degradation in RPA products
3. Agarose gel electrophoresis of RPA products
4. Cas12a cleavage of RPA products
5. Fluorescent detection of diluted Cas12a cleavage products

These experiments have been concluded before. For
detailed steps, refer to previous experiments.

2. **Test strip detection system**

1. RPA amplification
2. Protein degradation in RPA products
3. Agarose gel electrophoresis of RPA products
4. Cas12a cleavage of RPA products

For strip testing, FAM-ssDNA-Biotin was substituted
for ssDNA in the Cas12a cleavage reaction, compared to the fluorescent detection method.

5. Test strip analysis of Cas12a cleavage products

5 μL of Cas12a cleavage products were placed on the
sample pad of the test strip. The test strip was then placed in 70 μL of 1× PBS buffer (containing 0.01% Tween-20) for
chromatography. The appearance of a T line was observed within 2 min to determine the result.

These experiments have been concluded before. For
detailed steps, refer to previous experiments.

'
}]%}
{% block title %}experiments{% endblock %}
{% block lead %}
experiments
{% endblock %}

{% extends "layout.html" %}
{% set table\_data = [{
"title": "Plasmid construction",
"content": '

1. **Construction of the pET-28a-AsCpf1 plasmid**

To construct the plasmid pET-28a-AsCpf1, PCR
amplification of the AsCpf1 gene was performed. The pET-28a vector was then subjected to double digestion using BamH
I
and EcoR I enzymes. The gene fragment was ligated to the linearized vector using a homologous recombination method,
followed by transformation into BL21 competent cells. Single colonies were picked from the transformation plate and
verified by monoclonal PCR. The amplified products were further sequenced and analyzed by sequence alignment to
confirm successful construction of the recombinant plasmid.

![0767024dfc8a17a46e2bc004384bd773](https://static.igem.wiki/teams/6004/result/1.webp)

**Figure 1. The plasmid construction process of pET-28a-AsCpf1**

![](https://static.igem.wiki/teams/6004/result/2.webp)

**Figure 2. Results of pET-28a-AsCpf1 plasmid construction**

**Note:**

**A:** Agarose gel electrophoresis
results of PCR amplification of the AsCpf1 gene. M: 5K DNA Marker, N: negative control.

**B:** Double digestion results of
pET-28a using BamH I and EcoR I.

**C:** Culture plate for transforming
competent cells with recombinant plasmids.

**D:** Monoclonal PCR verification of
transformation results.

**E.** Sequence alignment results of
recombinant plasmid pET-28a-AsCpf1.

The construction process is illustrated in
**Figure 1**. The relevant results of pET-28a-AsCpf1 plasmid construction are shown in **Figure
2**. As depicted in **Figure 2A**, the PCR amplification of the AsCpf1 gene produced a single
band around 4000 bp, which aligns with the expected size of 3936 bp, confirming successful amplification of the
AsCpf1
gene. The double digestion results of the pET-28a vector are presented in **Figure 2B**. Upon
examination
of the digestion products, the presence of the expected band size and comparison with other lanes verified
successful
digestion. Subsequently, the recombinant plasmid was transformed into BL21 competent cells, and the appearance of
numerous single colonies on the transformation plate indicated a high transformation efficiency (**Figure
2C**). Transformation efficiency was further verified by monoclonal PCR, as shown in **Figure
2D**, where the expected band size was observed, confirming the presence of positive clones. Finally,
sequence analysis (**Figure 2E**) revealed that the amplified fragment matched the predicted sequence,
thus confirming the successful construction of the pET-28a-AsCpf1 plasmid.

2. **Construction of the pET-28a-c.520G plasmid**

To construct the plasmid pET-28a-c.520G, PCR
amplification of the c.520G gene fragment was performed. The pET-28a vector was then subjected to double digestion
using BamH I and EcoR I enzymes. The amplified gene fragment was ligated into the linearized vector using a
homologous
recombination method, followed by transformation into DH5α competent cells. Single colonies were picked from the
transformation plate and verified by monoclonal PCR. The amplified products were then sequenced and analyzed by
sequence alignment to confirm successful construction of the recombinant plasmid.

![](https://static.igem.wiki/teams/6004/result/3.webp)

**Figure 3. The plasmid construction process of pET-28a-c.520G**

***![](https://static.igem.wiki/teams/6004/result/4.webp)***

**Figure 4. Results of
pET-28a-c.520G plasmid
construction**

**Note:**

**A:** Agarose gel electrophoresis
results of PCR amplification of the c.520G gene. M: 2K DNA Marker, N: negative control.

**B:** Double digestion results of
pET-28a using BamH I and EcoR I.

**C:** Culture plate for transforming
competent cells with recombinant plasmids.

**D:** Monoclonal PCR verification of
transformation results.

**E:** Sequence alignment results of
recombinant plasmid pET-28a-c.520G.

The construction process is illustrated in
**Figure 3**. The relevant results of pET-28a-c.520G plasmid construction are shown in **Figure
4**. As depicted in **Figure 4A**, the PCR amplification of the c.520G gene produced a single
band around 362 bp, which aligns with the expected size of 362 bp, confirming successful amplification of the c.520G
gene. The double digestion results of the pET-28a vector are presented in **Figure 4B**. Upon
examination
of the digestion products, the presence of the expected band size and comparison with other lanes verified
successful
digestion. Subsequently, the recombinant plasmid was transformed into DH5α competent cells, and the appearance of
numerous single colonies on the transformation plate in **Figure 4C** indicated a high transformation
efficiency. Transformation efficiency was further verified by monoclonal PCR, as shown in **Figure
4D**,
where the expected band size was observed, confirming the presence of positive clones. Finally, sequence analysis
(**Figure 4E**) revealed that the amplified fragment matched the predicted sequence, thus confirming
the
successful construction of the pET-28a-c.520G plasmid.

3. **Construction of the pET-28a-c.520G>A plasmid**

For the construction of the pET-28a-c.520 G>A
plasmid, PCR amplification of the c .520G>A gene fragment was carried out. The pET-28a vector was then subjected
to
double digestion using BamH I and EcoR I enzymes to linearize it. The amplified c.520G>A fragment was ligated
into
the digested vector using a homologous recombination method, followed by transformation into DH5α competent cells.
After plating, individual colonies were selected, and monoclonal PCR was performed for initial screening. The cloned
products were sequenced and analyzed through sequence alignment to confirm the successful construction of the
recombinant plasmid.

***![](https://static.igem.wiki/teams/6004/result/5.webp)***

**Figure 5. The plasmid construction process
of
pET-28a-c.520G>A**

***![](https://static.igem.wiki/teams/6004/result/6.webp)***

**Figure 6. Results of pET-28a-c.520G>A
plasmid construction**

**Note:**

**A:** Agarose gel electrophoresis
showing the PCR amplification of the c.520G>A gene. M: 2K DNA Marker, N: negative control.

**B:** Double digestion results of
pET-28a vector using BamH I and EcoR I.

**C:** Transformation plate showing
DH5α
competent cells containing recombinant plasmids.

**D:** Monoclonal PCR results for
transformation verification.

**E:** Sequence alignment results
confirming the recombinant plasmid.

The process of plasmid construction is outlined in
**Figure 5**. The detailed results for the pET-28a-c.520G>A plasmid construction are presented in
**Figure 6**. As shown in **Figure 6A**, the PCR amplification of the c.520G>A gene
generated a single band around 362 bp, which corresponds with the expected fragment size, confirming successful gene
amplification. The results of the double digestion of the pET-28a vector are shown in **Figure 6B**,
where the digestion products reveal the presence of the expected fragment sizes, confirming proper digestion. The
transformation of the recombinant plasmid into DH5α cells resulted in the formation of numerous single colonies, as
illustrated in **Figure 6C**, indicating high transformation efficiency. The transformation was further
confirmed by monoclonal PCR, shown in **Figure 6D**, where the expected band size was detected,
confirming the presence of positive clones. Lastly, sequence analysis in **Figure 6E** confirmed that
the
sequence of the amplified c.520G>A fragment matched the expected sequence, validating the successful construction
of the pET-28a-c.520G>A plasmid.

4. **Construction of the pET-28a-c.6745C plasmid**

For the construction of the pET-28a-c.6745C
plasmid,
a PCR amplification was carried out to obtain the c.6745C gene fragment. The amplified product, which had a size of
310 bp, was inserted into a pET-28a vector that had been digested with BamH I and EcoR I enzymes. This was followed
by
ligation using a homologous recombination method, and the recombinant plasmid was then introduced into DH5α
competent
cells. After transformation, individual colonies were picked, and monoclonal PCR was used for verification. The
amplified PCR products were sequenced and compared to confirm the successful creation of the recombinant plasmid.

**![](https://static.igem.wiki/teams/6004/result/7.webp)**

**Figure 7. The construction procedure of the
pET-28a-c.6745C plasmid**

***![](https://static.igem.wiki/teams/6004/result/8.webp)***

**Figure 8. Results of pET-28a-c.6745C
plasmid
construction**

**Note**:

**A:** PCR amplification results of
the
c.6745C gene. M: 2K DNA Marker, N: negative control.

**B:** Double digestion of pET-28a
vector with BamH I and EcoR I enzymes.

**C:** Colony formation on the plate
after transformation with recombinant plasmids.

**D:** Monoclonal PCR verification of
the transformation results.

**E:** Sequence alignment of the
recombinant plasmid pET-28a-c.6745C.

The plasmid construction process is illustrated in
**Figure 7**. The results for the pET-28a-c.6745C plasmid construction are shown in **Figure
8**. In **Figure 8A**, PCR amplification of the c.6745C gene produced a single band around 310
bp, which matched the expected size, confirming the successful amplification of the c.6745C gene. **Figure
8B** shows the results of the double digestion of the pET-28a vector. The digestion products revealed the
expected band sizes, confirming the success of the digestion process. Following this, the recombinant plasmid was
transformed into DH5α competent cells, and **Figure 8C** shows the appearance of numerous colonies on
the
plate, indicating efficient transformation. Monoclonal PCR verification in **Figure 8D** confirmed the
presence of positive clones, as the expected band size was observed. Finally, the sequencing results shown in
**Figure 8E** indicated that the amplified fragment perfectly aligned with the expected sequence,
validating the successful construction of the pET-28a-c.6745C plasmid.

5. **Construction of the pET-28a-c.6745C>T plasmid**

For the creation of the pET-28a-c.6745C>T
plasmid,
the PCR amplification was performed using the c.6745C>T gene fragment. The amplified product, which had a size of
310 bp, was then inserted into a pET-28a vector previously digested with BamH I and EcoR I enzymes. The ligation
step
was completed using a homologous recombination method, and the recombinant plasmid was subsequently introduced into
DH5α competent cells. After transformation, individual colonies were selected and monoclonal PCR was conducted to
verify the presence of the desired insert. The PCR products were sequenced and aligned to ensure the successful
construction of the plasmid.

**![](https://static.igem.wiki/teams/6004/result/9.webp)**

**Figure 9. The construction process of the
pET-28a-c.6745C>T plasmid**

***![](https://static.igem.wiki/teams/6004/result/10.webp)***

**Figure 10. Results of the
pET-28a-c.6745C>T plasmid construction**

**Note:**

**A:** PCR amplification of the
c.6745C>T gene. M: 2K DNA Marker, N: negative control.

**B:** Double digestion of pET-28a
vector with BamH I and EcoR I.

**C:** Colony formation on the
transformation plate after recombinant plasmid introduction.

**D:** Monoclonal PCR verification of
the transformation.

**E:** Sequence alignment of the
recombinant plasmid pET-28a-c.6745C>T.

The plasmid construction process is shown in
**Figure 9**, with the corresponding results for the pET-28a-c.6745C>T plasmid construction
displayed
in **Figure 10**. As seen in **Figure 10A**, PCR amplification of the c.6745C>T gene
generated a distinct band at 310 bp, confirming the successful amplification of the gene. The results of the double
digestion of the pET-28a vector are presented in **Figure 10B**, where the expected band sizes were
observed, validating successful digestion. After the recombinant plasmid was transformed into DH5α competent cells,
**Figure 10C** demonstrates the appearance of several colonies on the transformation plate, confirming
effective transformation. The monoclonal PCR results shown in **Figure 10D** further supported the
presence of positive clones, as indicated by the expected band. Lastly, the sequence alignment results in
**Figure 10E** confirmed that the amplified fragment matched the anticipated sequence, confirming the
successful construction of the pET-28a-c.6745C>T plasmid.

'
},{
"title": "Protein expression of AsCpf1 (Cas12a) in pET-28a-AsCpf1-BL21 strain",
"content": '

1. **Optimization of IPTG-induced expression at 16 ℃**

To optimize the IPTG-induced expression of AsCpf1,
pET-28a-AsCpf1-BL21 acterial cultures were grown to an OD600 of 0.6, followed by IPTG induction at
concentrations of 0 mM, 0.2 mM, 0.5 mM, 0.8 mM, 1 mM, and 2 mM. The cultures were then incubated at 16 ℃ with
shaking
for 20 hours. After induction, protein was purified using a protein purification kit, and both crude and purified
protein samples were analyzed by SDS-PAGE.

![](https://static.igem.wiki/teams/6004/result/11.webp)

**Figure 11. IPTG-Induced Expression at 16 ℃
with different IPTG concentrations**

The electrophoresis results (**Figures 11A
and
11B**) showed clear overexpression of the target protein in the crude sample, with increasing IPTG
concentrations leading to stronger protein bands. After purification, the impurities in the crude protein were
almost
entirely removed, leaving predominantly the target protein. The intensity of the bands in both the crude and
purified
samples followed a consistent trend with the increasing IPTG concentration. The sample induced with 0.8 mM IPTG
exhibited the strongest band, both in the crude and purified forms, indicating that 0.8 mM IPTG is the optimal
concentration for protein expression at 16 ℃.

2. **Optimization of IPTG-induced expression at 37 ℃**

For higher-temperature expression, cultures were
induced with IPTG at concentrations of 0 mM, 0.2 mM, 0.5 mM, 0.8 mM, 1 mM, and 2 mM, after reaching an
OD600 of 0.6. The cultures were incubated at 37 ℃ with shaking for 3 hours. Afterward, protein was
purified
using the same protocol as the 16 ℃ induction, and the samples were analyzed by SDS-PAGE.

![](https://static.igem.wiki/teams/6004/result/12.webp)

**Figure 12. IPTG-Induced Expression at 37 ℃ with different IPTG concentrations**

The electrophoresis gel for the 37 ℃ induction
revealed weaker overall band intensities compared to the 16 ℃ induction (**Figure 12**). As the IPTG
concentration increased, the band intensity did not show significant changes. However, the samples induced with 0.5
mM
and 2 mM IPTG showed relatively stronger bands, suggesting these concentrations are optimal for expression at 37 ℃.
Although purification reduced some of the impurities, the overall protein expression at 37 ℃ was obviously weaker
than
at 16 ℃.

3. **Induction temperature optimization**
![](https://static.igem.wiki/teams/6004/result/13.webp)

**Figure 13. IPTG-Induced Expression at
different temperatures**

Comparing the results of IPTG induction at 16 ℃
(**Figure 13A**) and 37 ℃ (**Figure 13B**), it was observed that protein expression was
significantly higher at the lower temperature (16 ℃), especially at 0.8 mM IPTG. The overall band intensity at 16 ℃
was relatively high, and the purification process removed most impurities, leaving a clear target protein band. In
contrast, protein expression at 37 ℃ was weaker, even with higher IPTG concentrations, indicating that the lower
temperature (16 ℃) provides better conditions for AsCpf1 expression.

**Overall Conclusion**

Based on the results, induction at 16 ℃ with 0.8 mM
IPTG was chosen as the optimal expression condition, as it provided the strongest expression bands, indicating a
higher protein expression level.

'
},{
"title": "Establishment of an RPA-Cas12a detection platform",
"content": '

Purified Cas12a (AsCpf1) protein was quantified and
used to construct a dystrophic epidermolysis bullosa (DEB) detection platform targeting the c.520G>A and
c.6745C>T mutation sites in COL7A1. This detection platform includes the extraction of cell-free fetal DNA (cfDNA),
RPA amplification, Cas12a cleavage of the RPA products, and fluorescence detection of the Cas12a cleavage products.

It is noteworthy that to generate a significant
signal difference between the normal and mutant sequences, we systematically increased the number of mismatched bases
in the RPA primers. Different RPA primers were designed for RPA amplification. The optimal RPA primers were selected
based on the fluorescence signals generated by Cas12a cleavage. Subsequently, a reliable detection platform was
constructed using the selected primers. (The principle behind this design can be referenced in the "Our design"
section of the Project Description.)

1. **RPA reactions using different RPA primers**

   Conducted RPA reactions for c.520G (normal
   sequence),
   c.520G>A (mutant sequence), c.6745C (normal sequence), and c.6745C>T (mutant sequence), respectively. The
   primers used for the RPA reaction are: RPA-1M-c.520G>A-F/R, RPA-2M-c.520G>A-F/R, RPA-3M-c.520G>A-F/R,
   RPA-4M-c.520G>A-F/R, RPA-1M-c.6745C>T-F/R, RPA-2M-c.6745C>T-F/R, RPA-3M-c.6745C>T-F/R, and
   RPA-4M-c.6745C>T-F/R. RPA primer information is listed in the table below.

   **Table 1. RPA Primer information**

   |  |  |  |  |
   | --- | --- | --- | --- |
   | **Gene** | **Primer Name** | **Sequence (5’-3’)** | **Amplicon size** |
   | c .520G and c .520G>A | RPA-1M-c.520G>A-F | GACCTGGTGTCCCCAAGGTCTGCATCCTGA | 215 bp |
   | RPA-1M-c.520G>A-R | CAGTAGTGTCCTCAAGATGCTGAAGTCATTGA |
   | RPA-2M-c.520G>A-F | GAAGGGGCAGGGGGTCAAGCTATTTGCTGTC | 148 bp |
   | RPA-2M-c.520G>A-R | GGAAACGAGGGGCAGTAGTGTCCTCAAGATG |
   | RPA-3M-c.520G>A-F | GAAGGGGCAGGGGGTCAAGCTATTTGCTGAC | 148 bp |
   | RPA-3M-c.520G>A-R | GGAAACGAGGGGCAGTAGTGTCCTCAAGATG |
   | RPA-4M-c.520G>A-F | GAAGGGGCAGGGGGTCAAGCTATTTGCTCAC | 148 bp |
   | RPA-4M-c.520G>A-R | GGAAACGAGGGGCAGTAGTGTCCTCAAGATG |
   | c.6745C and c.6745C>T | RPA-1M-c.6745C>T-F | CTGACTGGACCTACTGGAGCTGTGGGACTTC | 164 bp |
   | RPA-1M-c.6745C>T-R | CTCCCTCTGTCTCCATCTTTTCCACTGGCAC |
   | RPA-2M-c.6745C>T-F | TTGTGGGTCCACAGGGGTCTCCAGGTTTGCCTGGC | 113 bp |
   | RPA-2M-c.6745C>T-R | AGGGCTCCCTCTGTCTCCATCTTTTCCACTG |
   | RPA-3M-c.6745C>T-F | TTGTGGGTCCACAGGGGTCTCCAGGTTTGCCTGCT | 113 bp |
   | RPA-3M-c.6745C>T-R | AGGGCTCCCTCTGTCTCCATCTTTTCCACTG |
   | RPA-4M-c.6745C>T-F | TTGTGGGTCCACAGGGGTCTCCAGGTTTGCCTCCT | 113 bp |
   | RPA-4M-c.6745C>T-R | AGGGCTCCCTCTGTCTCCATCTTTTCCACTG |

   1. **RPA product electrophoresis results for c.520G and c.520G>A**
   ![](https://static.igem.wiki/teams/6004/result/14.webp)

   **Figure 14. RPA product electrophoresis
   results for c.520G and c.520G>A**

   **Note:** A: c.520G>A

   In the electrophoresis image above, it can be
   observed that when using four different pairs of RPA primers, the target bands corresponding to the c.520G and
   c.520G>A templates are visible in the lanes of the RPA products, while the blank control group shows no bands
   (**Figure 14**). This indicates that both the normal and mutated sequences were successfully
   amplified,
   and there is no aerosol contamination. When using the RPA-1M-c.520G>A-F/R and RPA-2M-c.520G>A-F/R primers,
   the
   band intensity is brighter, whereas the bands corresponding to the RPA-3M-c.520G>A-F/R and
   RPA-4M-c.520G>A-F/R
   primers are relatively weaker. This is due to the latter primers introducing more mismatched sites, which can
   somewhat
   affect the RPA amplification efficiency

   2. **RPA product electrophoresis results for c.6745C and c.6745C>T**
   ![](https://static.igem.wiki/teams/6004/result/15.webp)

   **Figure 15. RPA product electrophoresis
   results for c.6745C and c.6745C>T**

   **Note:** B: c.6745C>T

   The RPA amplification results for the c.6745C
   and
   c.6745C>T templates are similar to those for the c.520G and c.6745C>T templates. As seen in the
   electrophoresis
   image above, when using four different pairs of RPA primers, the RPA products corresponding to the c.6745C and
   c.6745C>T templates show target bands, while no bands are observed in the blank control group (**Figure
   15**). This indicates that both the normal and mutated sequences were successfully amplified, and there
   is no
   aerosol contamination. When using the RPA-1M-c.6745C>T-F/R and RPA-2M-c.6745C>T-F/R primers, the band
   intensity
   is brighter, whereas the bands corresponding to the RPA-3M-c.6745C>T-F/R and RPA-4M-c.6745C>T-F/R primers
   are
   relatively weaker. This is due to the latter primers introducing more mismatched sites, which can somewhat
   affect the
   RPA amplification efficiency.
2. **Cas12a cleavage of RPA products**

   RPA amplification products of c.520G (normal
   sequence), c.520G>A (mutant sequence), c.6745C (normal sequence), and c.6745C>T (mutant sequence) using
   different primers were used in the Cas12a cleavage reaction. Among these, the c.520G and c.520G>A products were
   used for detecting the c.520G>A mutation site, while the c.6745C and c.6745C>T products were used for
   detecting
   the c.6745C>T mutation site.

   1. **Cas12a cleavage fluorescence results of RPA products corresponding to different RPA primers for
      c.520G and c.520G>A**

   ![](https://static.igem.wiki/teams/6004/result/16.webp)

   **Figure 16. Cas12a cleavage fluorescence results of RPA products corresponding to different RPA primers
   for
   c.520G and c.520G>A**

   Based on the results from real-time
   fluorescence
   monitoring and endpoint fluorescence detection (**Figure 16**), for the c.520G>A mutation site,
   using
   the RPA-1M-c.520G>A-F/R primers, both normal and mutant sequences exhibited strong fluorescence signals.
   Although
   the signal from the mutant sequence was stronger than that of the normal sequence, there was no significant
   difference
   between them, indicating that this primer pair does not meet the detection requirements. When using
   RPA-2M-c.520G>A-F/R, the normal sequence showed almost no fluorescence signal, while the mutant sequence
   exhibited
   a strong fluorescence signal, effectively distinguishing between the mutant and normal sequences. Therefore,
   this
   primer can be used for the detection of the c.520G>A mutation associated with DEB. However, no fluorescence
   signal
   was observed when using RPA-3M-c.520G>A-F/R and RPA-4M-c.520G>A-F/R, suggesting that the excessive
   mismatches
   introduced by these primers prevented the crRNA from recognizing the target DNA sequence, thus failing to
   activate
   Cas12a cleavage activity.

   In conclusion, RPA-2M-c.520G>A-F/R primers
   were
   selected for the detection of the c.520G>A mutation site.

   2. **Cas12a cleavage fluorescence results of RPA products corresponding to different RPA primers for
      c.6745C and c.6745C>T**

   ![](https://static.igem.wiki/teams/6004/result/17.webp)

   **Figure 17. Cas12a cleavage fluorescence results of RPA products corresponding to different RPA primers
   for
   c.6745C and c.6745C>T**

   For the c.6745C>T mutation site, real-time
   fluorescence monitoring and endpoint fluorescence detection results showed that using the
   RPA-1M-c.6745C>T-F/R,
   RPA-2M-c.6745C>T-F/R, and RPA-3M-c.6745C>T-F/R primers, both normal and mutant sequences exhibited strong
   fluorescence signals. Although the signal from the mutant sequence was stronger than that of the normal
   sequence,
   there was no significant difference between them, meaning these primers do not meet the detection requirements.
   However, when using RPA-4M-c.6745C>T-F/R, the normal sequence showed almost no fluorescence signal, while the
   mutant sequence showed a strong fluorescence signal, successfully distinguishing between the mutant and normal
   sequences (**Figure 17**). This primer can thus be used for detecting the c.6745C>T mutation
   site
   associated with DEB. Notably, when using the RPA-4M-c.6745C>T-F/R primer, the fluorescence signal from the
   mutant
   sequence significantly decreased. This is because the mismatches introduced by the RPA primer reduced the
   efficiency
   of crRNA recognition of the target DNA sequence, leading to a substantial decrease in Cas12a cleavage
   efficiency.

   In conclusion, RPA-4M-c.6745C>T-F/R primers
   were
   selected for the detection of the c.6745C>T mutation site.

**Conclusion**

A successful RPA-Cas12a detection platform was
established for the detection of dystrophic epidermolysis bullosa (DEB) mutations at the c.520G>A and
c.6745C>T
sites in COL7A1. After evaluating multiple RPA primer pairs, the optimal primers were selected based on their
ability
to generate distinct fluorescence signals following Cas12a cleavage. For the c.520G>A mutation, the
RPA-2M-c.520G>A-F/R primers were chosen as they provided a clear differentiation between the mutant and normal
sequences. Similarly, for the c.6745C>T mutation, the RPA-4M-c.6745C>T-F/R primers were selected, effectively
distinguishing the mutant sequence from the normal one. The constructed detection platform, incorporating cfDNA
extraction, RPA amplification, Cas12a cleavage, and fluorescence detection, is reliable for detecting these two
mutations associated with DEB.

'
},{
"title": "Time optimization of Cas12a cleavage reaction for the detection platforms",
"content": '

Building upon the results of the previous experiment,
the primers RPA-2M-c.520G>A-F/R and RPA-4M-c.6745C>T were selected for subsequent experiments. Due to varying
cleavage efficiencies of Cas12a at the two mutation sites, as well as the need to enhance detection performance, the
reaction time for Cas12a cleavage was optimized. Specifically, for the c.520G>A mutation, cleavage times (at 42 ℃)
were set at 0, 1, 2, 3, 4, and 5 minutes. For the c.6745C>T mutation, a real-time fluorescence device was used to
monitor the impact of enzyme cleavage time on detection performance, with a reaction time of 60 minutes.

1. **Optimization of Cas12a cleavage reaction time for c.520G and c.520G>A**

![](https://static.igem.wiki/teams/6004/result/18.webp)

**Figure 18. Optimization of Cas12a cleavage reaction time for c.520G and c.520G>A**

Based on the results from real-time fluorescence
monitoring and endpoint fluorescence detection, for the c.520G>A mutation site, a Cas12a cleavage reaction time
of
1 minute was sufficient to effectively distinguish between the mutant and normal sequences (with the mutant signal
being more than 3 times that of the normal sequence). As the reaction time increased, both the normal and mutant
sequence signals increased, with the signal from the mutant sequence showing a notably more significant enhancement,
while the normal sequence signal increased more slowly. Between reaction times of 1 to 5 minutes, the normal and
mutant sequences could be distinguished, indicating effective detection. Notably, at 3 minutes, the fluorescence
signal difference between the normal and mutant sequences was the most pronounced (**Figure 18**).
Given
that the sample concentration of the normal sequence was 109 copies/μL, a relatively high sample level,
the
lack of strong fluorescence signals from the normal sequence was due to the difficulty of matching the RPA-amplified
normal sequence with the crRNA, rather than low sample concentration.

Therefore, 3 minutes was determined to be the
optimal
Cas12a cleavage reaction time for detecting the c.520G>A mutation site.

2. **Optimization of Cas12a cleavage reaction time for c.6745C and c.6745C>T**

![](https://static.igem.wiki/teams/6004/result/19.webp)

**Figure 19. Optimization of Cas12a cleavage reaction time for c.6745C and c.6745C>T**

Based on the results from real-time fluorescence
monitoring, for the c.6745C>T mutation site, the efficiency of Cas12a cleavage of RPA-amplified products was
relatively low, which is consistent with the results observed during RPA primer screening. This is due to the
introduction of additional mismatched sites by the RPA primers, which reduced the recognition efficiency of crRNA
and
the cleavage efficiency of Cas12a (**Figure 19**). As the reaction time increased, the signal from the
mutant sequence continuously increased, reaching its maximum at 58 minutes, while the signal from the normal
sequence
remained unchanged over time.

Therefore, 60 minutes was determined to be the
optimal Cas12a cleavage reaction time for detecting the c.6745C>T mutation site.

**Conclusion:**

For the c.520G>A and c.6745C>T mutation
sites,
the RPA primers selected for the c.6745C>T mutation introduced more mismatched bases compared to the primers for
the c.520G>A mutation, resulting in a greater impact on crRNA recognition and Cas12a cleavage efficiency. As a
result, the fluorescence signal for c.6745C>T was relatively lower. However, based on the current results, the
detection of the c.6745C>T mutation site can still be successfully carried out.

'
},{
"title": "Feasibility analysis of the detection platforms",
"content": '

For the two mutation sites c.520G>A and
c.6745C>T, RPA amplification, Cas12a cleavage reaction, fluorescence detection, and test strip analysis were
performed on normal and mutant sequences under optimized conditions.

1. **Feasibility analysis for fluorescence detection platform**
![](https://static.igem.wiki/teams/6004/result/20.webp)

**Figure 20. Feasibility analysis for
fluorescence detection platform**

As shown in **Figure 20**, for the
c.520G>A and c.6745C>T mutation sites, the fluorescence signals from the mutant sequences were significantly
higher than those from the normal sequences, indicating that the optimized fluorescence detection system can
effectively distinguish between normal and mutant sequences. This detection system is feasible.

2. **Feasibility analysis for test strip detection platform**

![](https://static.igem.wiki/teams/6004/result/21.webp)

**Figure 21. Feasibility analysis for test strip detection platform**

As shown in **Figure 21**, for the
c.520G>A and c.6745C>T mutation sites, the test strips for the mutant sequences display both a control line (C
line) and a distinct test line (T line), while the normal sequences and blank controls only show the control line (C
line). This indicates that the optimized test strip detection system can effectively distinguish between normal and
mutant sequences, demonstrating the feasibility of this detection system.

**Conclusion:**

Both the fluorescence and test strip detection
platforms effectively distinguish between normal and mutant sequences at the c.520G>A and c.6745C>T mutation
sites. The optimized systems demonstrated clear differentiation, confirming their feasibility for mutation
detection.

'
},{
"title": "Molecular sensitivity analysis",
"content": '

For the two mutation sites c.520G>A and
c.6745C>T, to investigate the analytical sensitivity of the detection system, plasmid samples containing different
copy numbers of the mutated sequences were subjected to RPA amplification, Cas12a cleavage reaction, fluorescence
detection, and test strip analysis under optimized conditions.

The plasmid concentrations used were: 10⁸ copies/μL,
10⁵ copies/μL, 10⁴ copies/μL, 10³ copies/μL, 10² copies/μL, 10¹ copies/μL, and 10⁰ copies/μL.

1. **RPA product electrophoresis results for molecular sensitivity analysis for c.520G>A and
   c.6745C>T**
![](https://static.igem.wiki/teams/6004/result/22.webp)

**Figure 22. RPA product electrophoresis
results for molecular sensitivity analysis for c.520G>A and c.6745C>T**

In the electrophoresis gel (**Figure
22**), the RPA amplification products of samples 108, 106, and 105
copies/µL
showed bands that were consistent with the expected size. As the plasmid concentration decreased, the
electrophoretic
bands of the RPA products gradually weakened until they disappeared. This is due to the limited analytical capacity
of
RPA amplification combined with agarose gel electrophoresis. In summary, the appearance of the target band in the
high-concentration samples and the absence of bands in the blank control group indicate that the RPA amplification
reaction proceeded normally.

2. **Sensitivity analysis results of the fluorescence detection platform for c.520G>A**

![](https://static.igem.wiki/teams/6004/result/23.webp)

**Figure 23. Sensitivity analysis results of the fluorescence detection platform for c.520G>A**

Analysis of the fluorescence detection results for
the c.520G>A site revealed that as the plasmid concentration decreased, the fluorescence signal showed an overall
decreasing trend. The fluorescence value of the 103 copies/µL sample showed a significant difference
compared to the negative control, whereas the 102 copies/µL sample did not (**Figure 23**).
This indicates that the detection limit of the fluorescence detection system for the c.520G>A site is
103 copies/µL.

3. **Sensitivity analysis results of the test strip detection platform for c.520G>A**

![](https://static.igem.wiki/teams/6004/result/24.webp)

**Figure 24. Sensitivity analysis results of the test strip detection platform for c.520G>A**

Observing the test strip results in **Figure
24** for the c.520G>A site, it was found that as the plasmid concentration decreased, the strength of
the
T line on the test strip showed an overall decreasing trend. The 103 copies/µL sample displayed a clear T
line, while the 102 copies/µL sample did not. This indicates that the detection limit of the test strip
system for the c.520G>A site is 103 copies/µL.

4. **Sensitivity analysis results of the fluorescence detection platform for c.6745C>T**

![](https://static.igem.wiki/teams/6004/result/25.webp)

**Figure 25. Sensitivity analysis results of the fluorescence detection platform for c.6745C>T**

The fluorescence detection results for the
c.6745C>T site in **Figure 25** showed that as the plasmid concentration decreased, the fluorescence
signal overall exhibited a decreasing trend. The fluorescence value of the 105 copies/µL sample showed a
significant difference from the negative control, while the 104 copies/µL sample displayed some
fluorescence, but the data analysis revealed no significant difference compared to the negative control. This
suggests
that the detection limit of the fluorescence detection system for the c.6745C>T site is 105 copies/µL.

5. **Sensitivity analysis results of the test strip detection platform for c.6745C>T**

![](https://static.igem.wiki/teams/6004/result/26.webp)

**Figure 26. Sensitivity analysis results of the test strip detection platform for c.6745C>T**

Observing the test strip results for the
c.6745C>T
site, it was found that as the plasmid concentration decreased, the strength of the T line on the test strip also
showed a decreasing trend. As shown in **Figure 26,** the 104 copies/µL sample displayed a
clear T line, while the 103 copies/µL sample did not. This indicates that the detection limit of the test
strip system for the c.6745C>T site is 104 copies/µL.

**Conclusion:**

The molecular sensitivity analysis reveals that the
fluorescence detection system for the c.520G>A site has a detection limit of 10³ copies/µL, while the test strip
system has the same limit. For the c.6745C>T site, the fluorescence detection system has a limit of 10⁵
copies/µL,
and the test strip system has a limit of 10⁴ copies/µL.

'
},{
"title": "Random Sample Analysis",
"content": '

For the two target sites, c.520G>A and
c.6745C>T, standard plasmids containing both mutated and non-mutated sequences at varying concentrations
(≥104 copies/µL) were randomly mixed. A total of 12 random samples were prepared, and the constructed
method was used for analysis. All random samples were subjected to RPA amplification, Cas12a cleavage reaction,
fluorescence detection, and test strip analysis under optimized conditions. Positive and negative controls were also
set up in the experiment. PCR amplification is used to verify the presence of relevant genes (it cannot distinguish
whether they are mutant sequences).

1. **Random sample analysis for c.520G>A and c.6745C>T (fluorescence detection platform and test
   strip detection platform)**

**![](https://static.igem.wiki/teams/6004/result/27.webp)**

**Figure 27. Random sample analysis results
for
c.520G>A and c.6745C>T**

The results of the random sample analysis for the
c.520G>A and c.6745C>T targets are displayed in **Figure 27**. Both the fluorescence detection
and
test strip detection methods yielded consistent results, effectively distinguishing the presence or absence of
mutated
sequences. This indicates that the detection system can analyze actual samples.

2. **PCR validation of random samples**
![](https://static.igem.wiki/teams/6004/result/28.webp)

**Figure 28. PCR validation results of random
samples for c.520G>A and c.6745C>T**

Based on the comparison of PCR results (shown in
**Figure 28**) with fluorescence and test strip detection results, the following conclusions can be
drawn:

1. **COL7A1 c.520G>A**

Samples 2, 3, 5, 7, 8, 10, 11, and 12 contain the
COL7A1 c.520 near gene segment. Among these, samples 2, 3, 7, 10, and 11 contain the COL7A1 c.520G>A (mutated
sequence), while samples 5, 8, and 12 contain the COL7A1 c.520G (non-mutated sequence). Samples 1, 4, 6, and 9 do
not
contain the COL7A1 c.520 near gene segment.

2. **COL7A1 c.6745C>T**

Samples 2, 3, 5, 7, 10, 11, and 12 contain the
COL7A1
c.6745C near gene segment. Among these, samples 2, 3, 7, 10, and 11 contain the COL7A1 c.6745C>T (mutated
sequence), while samples 5 and 12 contain the COL7A1 c.6745C (non-mutated sequence). Samples 1, 4, 6, 8, and 9 do
not
contain the COL7A1 c.6745C near gene segment.

These results confirm that the detection system can
accurately identify both the presence of the targeted gene segments and the mutations associated with COL7A1.

'
}]%}
{% block title %}results{% endblock %}
{% block lead %}
results
{% endblock %}