Difference between revisions of "Part:BBa K2889003:Experience"

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===Applications of BBa_K2889003===
 
===Applications of BBa_K2889003===
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We constructed two plasmids pEGFP-miR-21-sponge-4s and PSB1C3-miR-21-sponge-4s. The plasmids of pEGFP-miR-21-sponge-4s and PSB1C3-miR-21-sponge-4s were used to monitor the expression of miR-21 in cells. And the plasmid of pSB1C3-miR-21-sponge-4s will be submitted to 2018IGEM.
  
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https://static.igem.org/mediawiki/parts/7/72/Electrophoresis_of_miR-21-sponge-4s_PCR_product.jpg
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In our previous project, we constructed two miR-21 sponge plasmid, pEGFP-miR-21-sponge-2s and pEGFP-miR-21-sponge-6s.Subsequently, we got two standard curves by transfected pEGFP-miR-21-sponge-2s and pEGFP-miR-21-sponge-6s with different concentration of pHAGE-pre-miR-21 to 293T cells and calculated the copy numbers of miR-21.However, we’d like to ascertain a preferable binding sites number contained in miRNA sponge as a sensitivity and specificity monitor this year.Hence, we constructed pEGFP-miR-21-sponge-4s.Firstly, we designed miR-21 sponges contains four miR-21 binding sites with 3-nt spacers for bulged sites based on the sequence of hsa-miR-21 according to the previ-ous study.Then we amplified miR-21-sponge-4s using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with restriction enzymes Xho I and Hind III. Finally, miR-21-sponge-4s fragments was ligated to pEGFP-c1 vector.To test the effect of miR-21 sponges as a monitor to detect the expression of miR-21, 293T cells were transfected with pEGFP-miR-21-sponge-4s with different concentra-tion of pHAGE-pre-miR-21. We used the plasmid of pHAGE-pre-miR-21 to quantify miR-21 expression and calculate the copy numbers of miR-21 by using the formula.Finally,We find that miR-21-sponge-4s is more sensitive than miR-21-sponge-2s and miR-21-sponge6s.
  
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https://static.igem.org/mediawiki/parts/0/01/The_effect_of_pEGFP-miR-21-sponge-4s_on_the_correlative_between_fluorescence_value_of_GFP_and_copy_numbers_of_miR-21.jpg
  
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First we amplified miR-21-sponge-4s using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with restriction enzymes EcoR I and Pst I
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https://static.igem.org/mediawiki/parts/c/c8/The_standard_curve_of_miR-21-sponge-4s.jpg
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https://static.igem.org/mediawiki/parts/5/5b/Digested_the_PSB1C3_vectors_with_EcoRI_and_PstI.jpeg
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We digested the PSB1C3 vectors with restriction enzymes EcoRI and PstI
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===User Reviews===
 
===User Reviews===

Latest revision as of 12:14, 9 October 2018


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K2889003

We constructed two plasmids pEGFP-miR-21-sponge-4s and PSB1C3-miR-21-sponge-4s. The plasmids of pEGFP-miR-21-sponge-4s and PSB1C3-miR-21-sponge-4s were used to monitor the expression of miR-21 in cells. And the plasmid of pSB1C3-miR-21-sponge-4s will be submitted to 2018IGEM.

In our previous project, we constructed two miR-21 sponge plasmid, pEGFP-miR-21-sponge-2s and pEGFP-miR-21-sponge-6s.Subsequently, we got two standard curves by transfected pEGFP-miR-21-sponge-2s and pEGFP-miR-21-sponge-6s with different concentration of pHAGE-pre-miR-21 to 293T cells and calculated the copy numbers of miR-21.However, we’d like to ascertain a preferable binding sites number contained in miRNA sponge as a sensitivity and specificity monitor this year.Hence, we constructed pEGFP-miR-21-sponge-4s.Firstly, we designed miR-21 sponges contains four miR-21 binding sites with 3-nt spacers for bulged sites based on the sequence of hsa-miR-21 according to the previ-ous study.Then we amplified miR-21-sponge-4s using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with restriction enzymes Xho I and Hind III. Finally, miR-21-sponge-4s fragments was ligated to pEGFP-c1 vector.To test the effect of miR-21 sponges as a monitor to detect the expression of miR-21, 293T cells were transfected with pEGFP-miR-21-sponge-4s with different concentra-tion of pHAGE-pre-miR-21. We used the plasmid of pHAGE-pre-miR-21 to quantify miR-21 expression and calculate the copy numbers of miR-21 by using the formula.Finally,We find that miR-21-sponge-4s is more sensitive than miR-21-sponge-2s and miR-21-sponge6s.

The_effect_of_pEGFP-miR-21-sponge-4s_on_the_correlative_between_fluorescence_value_of_GFP_and_copy_numbers_of_miR-21.jpg

The_standard_curve_of_miR-21-sponge-4s.jpg

User Reviews

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