Difference between revisions of "Part:BBa K4712086"
 
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<h1> Protocol
+
Protocol:
−
 
+
 
Apparatus:
 
Apparatus:
 
Thermal Cycler, Centrifuge, Fluorescence Quantitative PCR Instrument (Ya Rui), 2mL reaction tube, Pipette and Pipette Tip
 
Thermal Cycler, Centrifuge, Fluorescence Quantitative PCR Instrument (Ya Rui), 2mL reaction tube, Pipette and Pipette Tip
−
 
 
Materials:
 
Materials:
−
1. DNA Isothermal Amplification Kit (EZ-Life Biotechnology)
+
1. DNA Isothermal Amplification Reagent Kit (EasyGene Biotechnology)
 
2. ddH2O
 
2. ddH2O
−
3. Thermostatic amplification specific primers:
+
3. Isothermal Amplification Specific Primers:  
−
  INFB-F1、 INFB-R1
+
−
  INFB-F2、 INFB-R2
+
−
  INFB-F3、 INFB-R3
+
−
  INFB-F4、 INFB-R4
+
−
  INFB-F5、 INFB-R5
+
−
  H1N1-F6、H1N1-R6
+
−
  H1N1-F7、H1N1-R7
+
−
  H1N1-F8、H1N1-R8
+
−
4. DL500 marker
+
−
5. Test sample: DNA Template（pUC57-M1）Concentration：1000cps/μL
+
−
Storage: -20°C
+
  
 +
<table>
 +
<thead>
 +
  <tr>
 +
    <th>H5N1-F1</th>
 +
    <th>H5N1-R1</th>
 +
    <th>H5N1-F2</th>
 +
    <th>H5N1-R2</th>
 +
    <th>H5N1-F3</th>
 +
    <th>H5N1-R3</th>
 +
    <th>H5N1-F4</th>
 +
    <th>H5N1-R4</th>
 +
  </tr>
 +
</thead>
 +
<tbody>
 +
  <tr>
 +
    <td>H5N6-F1</td>
 +
    <td>H5N6-R1</td>
 +
    <td>H5N6-F2</td>
 +
    <td>H5N6-R2</td>
 +
    <td>H5N6-F3</td>
 +
    <td>H5N6-R3</td>
 +
    <td>H5N6-F4</td>
 +
    <td>H5N6-R4</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Mtu-F1</td>
 +
    <td>Mtu-R1</td>
 +
    <td>Mtu-F2</td>
 +
    <td>Mtu-R2</td>
 +
    <td>Mtu-F3</td>
 +
    <td>Mtu-R3</td>
 +
    <td>Mtu-F4</td>
 +
    <td>Mtu-R4</td>
 +
  </tr>
 +
  <tr>
 +
    <td>NME-F1</td>
 +
    <td>NME-R1</td>
 +
    <td>NME-F2</td>
 +
    <td>NME-R2</td>
 +
    <td>NME-F3</td>
 +
    <td>NME-R3</td>
 +
    <td>NME-F4</td>
 +
    <td>NME-R4</td>
 +
  </tr>
 +
  <tr>
 +
    <td>SARS-CoV-F1</td>
 +
    <td>SARS-CoV-R1</td>
 +
    <td>SARS-CoV-F2</td>
 +
    <td>SARS-CoV-R2</td>
 +
    <td>SARS-CoV-F3</td>
 +
    <td>SARS-CoV-R3</td>
 +
    <td>SARS-CoV-F4</td>
 +
    <td>SARS-CoV-R4</td>
 +
  </tr>
 +
  <tr>
 +
    <td>SARS-CoV-F5</td>
 +
    <td>SARS-CoV-R5</td>
 +
    <td>SARS-CoV-F6</td>
 +
    <td>SARS-CoV-R6</td>
 +
    <td>SARS-CoV-F7</td>
 +
    <td>SARS-CoV-R7</td>
 +
    <td>SARS-CoV-F8</td>
 +
    <td>SARS-CoV-R8</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Cd-F1</td>
 +
    <td>Cd-R1</td>
 +
    <td>Cd-F2</td>
 +
    <td>Cd-R2</td>
 +
    <td>Cd-F3</td>
 +
    <td>Cd-R3</td>
 +
    <td></td>
 +
    <td></td>
 +
  </tr>
 +
  <tr>
 +
    <td>SP-F1</td>
 +
    <td>SP-R1</td>
 +
    <td>SP-F2</td>
 +
    <td>SP-R2</td>
 +
    <td>SP-F3</td>
 +
    <td>SP-R3</td>
 +
    <td>SP-F4</td>
 +
    <td>SP-R4</td>
 +
  </tr>
 +
  <tr>
 +
    <td>HAdV-B-F1</td>
 +
    <td>HAdV-B-R1</td>
 +
    <td>HAdV-B-F2</td>
 +
    <td>HAdV-B-R2</td>
 +
    <td>HAdV-B-F3</td>
 +
    <td>HAdV-B-R3</td>
 +
    <td>HAdV-B-F4</td>
 +
    <td>HAdV-B-R4</td>
 +
  </tr>
 +
  <tr>
 +
    <td>HAdV-C-F1</td>
 +
    <td>HAdV-C-R1</td>
 +
    <td>HAdV-C-F2</td>
 +
    <td>HAdV-C-R2</td>
 +
    <td>HAdV-C-F3</td>
 +
    <td>HAdV-C-R3</td>
 +
    <td></td>
 +
    <td></td>
 +
  </tr>
 +
</tbody>
 +
</table>
 +
 +
4. DL500 marker
 +
5. Test Sample: DNA Template（pUC57-M1）Concentration：1000cps/μL
 +
Storage: -20℃
 
Methods:
 
Methods:
 
1. For a 20μL reaction system:
 
1. For a 20μL reaction system:
 +
 
<table>
 
<table>
 
<thead>
 
<thead>
Line 71: Line 169:
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td>H2O</td>
+
     <td>ddH2O</td>
 
     <td></td>
 
     <td></td>
 
     <td>To 18</td>
 
     <td>To 18</td>
Line 82: Line 180:
 
</tbody>
 
</tbody>
 
</table>
 
</table>
−
2. Gently tap multiple times to mix the ingredient, and then centrifuge briefly (mix gently, avoiding vigorous shaking or vortexing).
+
 
−
3. Incubate at 37 ℃ for 20 minutes.
+
2. Gently tap to mix several times, briefly centrifuge, repeat 3 times (mix gently to avoid vigorous vortexing).
−
4. After heating at 65 ℃ for 10 minutes, the adhesive will run off.
+
3. Incubate at 37°C for 20 minutes.
 +
4. After heating at 65°C for 10 minutes, proceed to gel electrophoresis.
 +
Results:
 +
 
 +
Results:
 +
 
 +
https://static.igem.wiki/teams/4712/wiki/result/fig7g.png
 +
 
 +
(g) Electrophoresis of RPA Primer Screening for Corynebacterium diphtheriae (Cd)
 +
 
 +
https://static.igem.wiki/teams/4712/wiki/result/fig9d.png
 +
 
 +
The figure above correspond to the fluorescence intensity of crRNA targeting Severe acute respiratory Corynebacterium diphtheriae after CRISPR reaction under Bright and UV illumination. The figure utilize pseudocolor to facilitate analysis using software (Image Lab 6). No crRNA is added into negative control. The concentration of DNA template is 10nM/L.
 +
 
 +
https://static.igem.wiki/teams/4712/wiki/result/fig8d.png
 +
 
 +
The linear graphs sequentially correspond to the efficiency verification of crRNAs targeting the DNA sequences of Corynebacterium diphtheriae pathogen. No crRNA is added into negative control. The concentration of DNA template is 10nM/L.
 +
 
 +
https://static.igem.wiki/teams/4712/wiki/result/fig10e-right.png
 +
 
 +
Linear graphs and figure correspond to the fluorescence intensity of crRNAs targeting Corynebacterium diphtheriae pathogen after one-tube reaction of RPA and CRISPR under Bright and UV illumination. The figures utilize pseudocolor to facilitate analysis using software (Image Lab 6). No crRNA is added into negative control. The concentration of DNA template is 10nM/L.
 +
 
 +
Machine Learning
 +
https://static.igem.wiki/teams/4712/wiki/engineering/engineering/fig1.png
 +
 
 +
In the initial phase, we trained the model with a limited dataset of 74 data points, followed by an evaluation that proved that the support vector machine model fits into the evaluation.
 +
 
 +
Subsequently, during the second round, our focus shifted to fine-tuning the SVM model parameters. Notably, this round of training also relied on the same 74 data points. It is pertinent to mention that both the first and second rounds of training and evaluation employed randomly partitioned datasets.
 +
 
 +
In the third round, our model made predictions for the performance of 40 entirely new primers, which were unlabeled. The practical utility of these predictions was validated through experimental verification, confirming the high accuracy and reliability of our model. The alignment between the predicted class and the experimental class was particularly strong.
 +
 
 +
This approach effectively underscores the potential of machine learning to reduce the number of experiments required.

Latest revision as of 13:37, 12 October 2023


Cd-F1

The primers were designed using NCBI BLAST and SanpGene to achieve efficient and specific amplification. This primer in RPA serves as the initial binding point for the amplification process, ensuring the specificity of the reaction by targeting the desired Corynebacterium diphtheria DNA or RNA sequences. The primers provided data for mathematical modeling for further primer design.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Protocol: Apparatus: Thermal Cycler, Centrifuge, Fluorescence Quantitative PCR Instrument (Ya Rui), 2mL reaction tube, Pipette and Pipette Tip Materials: 1. DNA Isothermal Amplification Reagent Kit (EasyGene Biotechnology) 2. ddH2O 3. Isothermal Amplification Specific Primers:

<thead> </thead> <tbody> </tbody>
H5N1-F1 H5N1-R1 H5N1-F2 H5N1-R2 H5N1-F3 H5N1-R3 H5N1-F4 H5N1-R4
H5N6-F1 H5N6-R1 H5N6-F2 H5N6-R2 H5N6-F3 H5N6-R3 H5N6-F4 H5N6-R4
Mtu-F1 Mtu-R1 Mtu-F2 Mtu-R2 Mtu-F3 Mtu-R3 Mtu-F4 Mtu-R4
NME-F1 NME-R1 NME-F2 NME-R2 NME-F3 NME-R3 NME-F4 NME-R4
SARS-CoV-F1 SARS-CoV-R1 SARS-CoV-F2 SARS-CoV-R2 SARS-CoV-F3 SARS-CoV-R3 SARS-CoV-F4 SARS-CoV-R4
SARS-CoV-F5 SARS-CoV-R5 SARS-CoV-F6 SARS-CoV-R6 SARS-CoV-F7 SARS-CoV-R7 SARS-CoV-F8 SARS-CoV-R8
Cd-F1 Cd-R1 Cd-F2 Cd-R2 Cd-F3 Cd-R3
SP-F1 SP-R1 SP-F2 SP-R2 SP-F3 SP-R3 SP-F4 SP-R4
HAdV-B-F1 HAdV-B-R1 HAdV-B-F2 HAdV-B-R2 HAdV-B-F3 HAdV-B-R3 HAdV-B-F4 HAdV-B-R4
HAdV-C-F1 HAdV-C-R1 HAdV-C-F2 HAdV-C-R2 HAdV-C-F3 HAdV-C-R3

4. DL500 marker 5. Test Sample: DNA Template（pUC57-M1）Concentration：1000cps/μL Storage: -20℃ Methods: 1. For a 20μL reaction system:

<thead> </thead> <tbody> </tbody>
Reagent Stock Concentration Volume Added（μL）
Forward Primer 10μM 1
Reverse Primer 10μM 1
Rehydration Buffer (2X) 10
DNA Template 10nM/L 2
ddH2O To 18
Starter (10X) 2

2. Gently tap to mix several times, briefly centrifuge, repeat 3 times (mix gently to avoid vigorous vortexing). 3. Incubate at 37°C for 20 minutes. 4. After heating at 65°C for 10 minutes, proceed to gel electrophoresis. Results:

Results:

fig7g.png

(g) Electrophoresis of RPA Primer Screening for Corynebacterium diphtheriae (Cd)

fig9d.png

The figure above correspond to the fluorescence intensity of crRNA targeting Severe acute respiratory Corynebacterium diphtheriae after CRISPR reaction under Bright and UV illumination. The figure utilize pseudocolor to facilitate analysis using software (Image Lab 6). No crRNA is added into negative control. The concentration of DNA template is 10nM/L.

fig8d.png

The linear graphs sequentially correspond to the efficiency verification of crRNAs targeting the DNA sequences of Corynebacterium diphtheriae pathogen. No crRNA is added into negative control. The concentration of DNA template is 10nM/L.

fig10e-right.png

Linear graphs and figure correspond to the fluorescence intensity of crRNAs targeting Corynebacterium diphtheriae pathogen after one-tube reaction of RPA and CRISPR under Bright and UV illumination. The figures utilize pseudocolor to facilitate analysis using software (Image Lab 6). No crRNA is added into negative control. The concentration of DNA template is 10nM/L.

Machine Learning fig1.png

In the initial phase, we trained the model with a limited dataset of 74 data points, followed by an evaluation that proved that the support vector machine model fits into the evaluation.

Subsequently, during the second round, our focus shifted to fine-tuning the SVM model parameters. Notably, this round of training also relied on the same 74 data points. It is pertinent to mention that both the first and second rounds of training and evaluation employed randomly partitioned datasets.

In the third round, our model made predictions for the performance of 40 entirely new primers, which were unlabeled. The practical utility of these predictions was validated through experimental verification, confirming the high accuracy and reliability of our model. The alignment between the predicted class and the experimental class was particularly strong.

This approach effectively underscores the potential of machine learning to reduce the number of experiments required.