Difference between revisions of "Part:BBa K4308000"

(→‎Characterization)
 
(14 intermediate revisions by the same user not shown)
Line 3: Line 3:
 
<partinfo>BBa_K4308000 short</partinfo>
 
<partinfo>BBa_K4308000 short</partinfo>
  
−
Long intergenic non-protein coding RNA 857 (LINC00857) is a genetic molecule involved in cancer biology, which is identified as one of the most upregulated LINCRNAs in lung cancer. It could lead to resistance to platinum&#8208;induced apoptosis by affecting mRNA stability or promoting its translation. Thus, we chose LINC00857 as a novel prognostic and cancer predictive biomarker. We use this part as the forward primer to conduct recombinase polymerase amplification (RPA) of LINC00857, along with its reverse primer (<partinfo>BBa_K4308001</partinfo>).
+
Long intergenic non-protein coding RNA 857 (LINC00857) is a genetic molecule involved in cancer biology, which is identified as one of the most upregulated LINCRNAs in lung cancer. It could lead to resistance to platinum&#8208;induced apoptosis by affecting mRNA stability or promoting its translation. Thus, we chose LINC00857 as a novel prognostic and cancer predictive biomarker. We use this part as the forward primer to conduct reverse transcription-recombinase polymerase amplification (RT-RPA) of LINC00857, along with its reverse primer (<partinfo>BBa_K4308001</partinfo>).
−
 
+
  
 
===Usage and Biology===
 
===Usage and Biology===
  
−
In our project, this primer (<partinfo>BBa_K4308000</partinfo>) was used along with its reverse primer (<partinfo>BBa_K4308001</partinfo>) for the amplification reactions of LINC00857, in order to perform isothermal amplification reactions with recombinase polymerase amplification (RPA). Using this method, we were able to detect quantities as low as <var>10<sup>-9</sup></var> nanograms during 30 minutes. The expected amplified product length is about 150 bp.  
+
In our project, this primer (<partinfo>BBa_K4308000</partinfo>) was used along with its reverse primer (<partinfo>BBa_K4308001</partinfo>) for the amplification reactions of LINC00857, in order to perform isothermal amplification reactions with RT-RPA. Using this method, we were able to detect quantities as low as <var>10<sup>-9</sup></var> nanograms during 30 minutes. The expected amplified product length is about 150 bp.  
  
 +
<html>
 +
    <figure style="display: flex; justify-content: center; align-items: center;">
 +
        <img src="https://static.igem.wiki/teams/4308/wiki/static/imgs/parts-registry/bba-k4308000/figure1.gif" width="60%" style="float:center;">
 +
        <figcaption>
 +
        <p style="font-size:1rem">
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
</html>
  
−
===<h1>Characterization</h1>===
 
−
<p><b>1.Proof of the expression</b></p>
 
−
<p>After RPA amplification, we carried out 3% agarose gel electrophoresis to verify the amplification results. As can be seen in Figure 1, compared with negative control, our product has obvious bands, which also confirms the validity of the primer.</p>
 
 
<html>
 
<html>
 
     <figure style="display: flex; justify-content: center; align-items: center;">
 
     <figure style="display: flex; justify-content: center; align-items: center;">
−
         <img src="图片链接" width="40%" style="float:center;">
+
         <img src="https://static.igem.wiki/teams/4308/wiki/static/imgs/parts-registry/bba-k4308000/fig-2.png" width="80%" style="float:center;">
 
         <figcaption>
 
         <figcaption>
 
         <p style="font-size:1rem">
 
         <p style="font-size:1rem">
Line 23: Line 28:
 
     </figure>
 
     </figure>
 
</html>
 
</html>
−
<p style="text-align:center"><b>Figure.1 3% agarose gel electrophoresis to verify the amplification</b></p>
+
<p style="text-align:center"><b>Figure.1 Schematic diagram of RT-RPA.</b></p>
  
  
 +
===<h1>Characterization</h1>===
 +
 +
<p><b>1. Experimental verification of RT-RPA</b></p>
 +
<p>The results of the above RPA pre-experiment were better, so we replaced our target gene LINC00857 for reverse transcription-recombinase polymerase amplification (RT-RPA). After RT-RPA amplification, we carried out 3% agarose gel electrophoresis to verify the amplification results. As can be seen in Figure 2, compared with negative control, our product has obvious bands, which also confirms the validity of the primer.</p>
 +
<html>
 +
    <figure style="display: flex; justify-content: center; align-items: center;">
 +
        <img src="https://static.igem.wiki/teams/4308/wiki/static/imgs/parts-registry/bba-k4308000/rt-rpa.png" width="60%" style="float:center;">
 +
        <figcaption>
 +
        <p style="font-size:1rem">
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
</html>
 +
<p style="text-align:center"><b>Figure.2 3% agarose gel electrophoresis to verify the amplification.</b> <i>Lane 1: Target gene ( 10<sup>1</sup> copies); Lane 2 : Target gene ( 10<sup>2</sup> copies); Lane 3 : Target gene ( 10<sup>3</sup> copies); Lane 4 : Target gene ( 10<sup>4</sup> copies). </i></p>
  
  

Latest revision as of 23:07, 11 October 2022


Forward Primer for RPA of LINC00857

Long intergenic non-protein coding RNA 857 (LINC00857) is a genetic molecule involved in cancer biology, which is identified as one of the most upregulated LINCRNAs in lung cancer. It could lead to resistance to platinum‐induced apoptosis by affecting mRNA stability or promoting its translation. Thus, we chose LINC00857 as a novel prognostic and cancer predictive biomarker. We use this part as the forward primer to conduct reverse transcription-recombinase polymerase amplification (RT-RPA) of LINC00857, along with its reverse primer (BBa_K4308001).

Usage and Biology

In our project, this primer (BBa_K4308000) was used along with its reverse primer (BBa_K4308001) for the amplification reactions of LINC00857, in order to perform isothermal amplification reactions with RT-RPA. Using this method, we were able to detect quantities as low as 10-9 nanograms during 30 minutes. The expected amplified product length is about 150 bp.

Figure.1 Schematic diagram of RT-RPA.


Characterization

1. Experimental verification of RT-RPA

The results of the above RPA pre-experiment were better, so we replaced our target gene LINC00857 for reverse transcription-recombinase polymerase amplification (RT-RPA). After RT-RPA amplification, we carried out 3% agarose gel electrophoresis to verify the amplification results. As can be seen in Figure 2, compared with negative control, our product has obvious bands, which also confirms the validity of the primer.

Figure.2 3% agarose gel electrophoresis to verify the amplification. Lane 1: Target gene ( 101 copies); Lane 2 : Target gene ( 102 copies); Lane 3 : Target gene ( 103 copies); Lane 4 : Target gene ( 104 copies).


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]