Difference between revisions of "Part:BBa K4223003"
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<h3>Description</h3>
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<h3>Experience</h3>
 
In order to provide data support for false positives, we introduced 13 DNA sequences, including 1 Target and 12 DNA sequences containing odd locus single base mutations. The specific mutation sites are shown in Figure 1.
 
In order to provide data support for false positives, we introduced 13 DNA sequences, including 1 Target and 12 DNA sequences containing odd locus single base mutations. The specific mutation sites are shown in Figure 1.
 
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<img src="C:\Users\yu\Desktop\Parts\Transcription system of target RNA for Cas13a1\Figure 1" alt="">
 
<img src="C:\Users\yu\Desktop\Parts\Transcription system of target RNA for Cas13a1\Figure 1" alt="">
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<h3>Experience</h3>
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The iGEM 2021 NEFU_China introduced the single stand sequence of G-quadruplex with ssDNA into the circular DNA template. This was achieved by primer incubation, followed by the addition of Phi29 DNA polymerase and dNTPs into the RCA buffer; hemin was then added into the reaction system followed by incubation and ABTS addition. With the presence of the viral RNA sequence in the initial sample, the G-quadruplex will be amplified and the reaction solution will change to green color<sup>[1-2]</sup>.
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'''Figure 1. Single nucleotide polymorphisms(SNPS) at odd loci in 12 different DNA and Target'''
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<img src="https://2021.igem.org/wiki/images/c/c1/T--NEFU_China--NEFU-xiguangdu.png" style="width:40%;height=40%; "></img>
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Figure 1. Absorbance curves of different concentrations of G-quadruplex.
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In order to put our detection device into actual application, we explored the relationship between absorbance and the primer amount according to the detectable color range, and finally determined the required sample size through the modeling. Therefore, we designed a gradient of primer concentrations for RCA and chromogenic reaction, and obtained the results.
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<img src="https://2021.igem.org/wiki/images/7/75/T--NEFU_China--RCA_con.png" style="width:30%;height=30%; "></img>
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Figure 2. Results of G-quadruplex electrophoresis by Phi29-mediated RCA.
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<img src="https://2021.igem.org/wiki/images/4/42/T--NEFU_China--Results-21.png" style="width:45%;height=60%; "></img>
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Figure 3. Chromogenic reaction of Phi29-mediated RCA products. | 1. Positive control with a synthesized G-quadruplex sequence. 2. Products of Phi29-mediated RCA. 3. Reaction without primers. 4. Reaction without template. 5. Reaction without Phi29 polymerase.  6. Reaction without T4 DNA ligase.
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<img src="https://2021.igem.org/wiki/images/d/db/T--NEFU_China--Results-22.png" style="width:45%;height=45%; "></img>
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Figure 4. Absorbance at 420nm of the chromogenic reaction of the Phi29-mediiated RCA products.
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The iGEM 2021 NEFU_China used purified Phi29 polymerase to carry out RCA for the amplification the G-quadruplex template. Based on the DNA agarose electrophoresis, the RCA products could be steadily detected in the experimental group but not in the controls (Figure 2). Meanwhile, the color change could also be observed in the experimental group versus the controls (Figure 3), and their absorbance at 420 nm was also quantified (Figure 4).  From the results of these three experiments, we conclude that Phi29 can efficiently amplify the G-quadruplex sequence through the RCA reaction and the amplified product can generate chromogenic reaction.
 
 
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Through the recognition and cleavage of the artificially designed RNA by Cas13a1 protein, the trans-cleavage activity was displayed, and the resulting fluorescence signal was revealed as Relative fluorescence Unit (RFU). We could observe that each detected sequence showed different fluorescence signal intensities, and the signal intensities of M20 and M7 were significantly greater than the target sequences. This indicates that false positives do exist, and different mutation sites have different effects on the specificity of the detection.
 
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<h3>References</h3>
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'''Figure 2. Detection results of Cas13a protein for each sequence (revealed by relative fluorescence unit RFU)'''
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[1] R. Connelly, C. Verduzco, et al., Toward a rational approach to design split G-quadruplex probes, ACS Chem. Bio, 10:1021, 2019.
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'''Figure 3. Schematic representation of the Cas13a-sgRNA-Target complex'''
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[2] T. Yoshimura, S. Arikado and S. Ohuchi. Estimation of DNA polymerase for improvement of rolling circle amplification, Oxford University Press, 820-8502, 2006.
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<span class='h3bb'>Sequence and Features</span>
 
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Revision as of 16:19, 20 September 2022


Transcription system of target RNA for LbCas13a

Cas13a tolerates one or two mismatches between crRNA and the target sequence, which will result in a greatly reduced cleavage efficiency of Cas13a protein, and this wrong cleavage will also lead to "false positives" in the detection.
In order to confirm the occurrence of false positives, Hainanu-China designed a series of sequences with single nucleotide polymorphisms (SNPS) based on the target RNA, which were detected by fluorescence reporter.
It should be noted that fluorescence was generated by trans-cleavage of ssRNA cleavage Reporter after Cas13a recognized and cleaved the target RNA.


Experience

In order to provide data support for false positives, we introduced 13 DNA sequences, including 1 Target and 12 DNA sequences containing odd locus single base mutations. The specific mutation sites are shown in Figure 1.

<img src="C:\Users\yu\Desktop\Parts\Transcription system of target RNA for Cas13a1\Figure 1" alt="">

Figure 1. Single nucleotide polymorphisms(SNPS) at odd loci in 12 different DNA and Target

Through the recognition and cleavage of the artificially designed RNA by Cas13a1 protein, the trans-cleavage activity was displayed, and the resulting fluorescence signal was revealed as Relative fluorescence Unit (RFU). We could observe that each detected sequence showed different fluorescence signal intensities, and the signal intensities of M20 and M7 were significantly greater than the target sequences. This indicates that false positives do exist, and different mutation sites have different effects on the specificity of the detection.
Figure 2. Detection results of Cas13a protein for each sequence (revealed by relative fluorescence unit RFU) Figure 3. Schematic representation of the Cas13a-sgRNA-Target complex


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 100
  • 1000
    COMPATIBLE WITH RFC[1000]