Difference between revisions of "Part:BBa K2889000:Design"

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IL7-AS-S2 fragments were ligated to PSB1C3 vector. Then we selected the positive clones by PCR and sequencing (Fig 3).
 
IL7-AS-S2 fragments were ligated to PSB1C3 vector. Then we selected the positive clones by PCR and sequencing (Fig 3).
 
https://static.igem.org/mediawiki/parts/f/f9/Verify_pSB1C3-IL7-AS-S2_by_sequencing.jpeg
 
https://static.igem.org/mediawiki/parts/f/f9/Verify_pSB1C3-IL7-AS-S2_by_sequencing.jpeg
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The secondary structure of IL7-AS-S2(Fig 4).
 
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https://static.igem.org/mediawiki/parts/a/a1/IL7-AS-S2.jpg
 
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Cell migration is a central process in the development and maintenance of tumor. We cloned the full length of IL7-AS, two truncated sequences of IL7-AS (IL7-AS-S1 and IL7-AS-S2) into PCDNA3.1(Fig 5) and transfected these plasmids to 786-O cells. Through in vitro scratch wound healing assay, overexpression of IL7-AS-S2 promoted cell migration of 786-O cells(Fig 6 and 7).These results suggested that IL7-AS-S2 contain key structural domains.
 
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https://static.igem.org/mediawiki/parts/9/95/Scheme_of_three_IL7-AS_isoforms.jpg
 
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https://static.igem.org/mediawiki/parts/8/8c/IL7-AS-AS2.jpg
 
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https://static.igem.org/mediawiki/parts/a/ad/IL7-AS-S2-cell_migration.jpg
 
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In order to investigate whether cell migration induced by IL7-AS-S2 depends on the amount of IL7-AS-S2 transfection. We transfected the different concentration of IL7-AS-S2 into 786-0 cells. But the results showed no significant difference.(Fig 8 and 9)
 
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https://static.igem.org/mediawiki/parts/6/6a/Different_concentration_of_IL7-AS-S2.jpg
 
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https://static.igem.org/mediawiki/parts/b/bb/Different_concentration_of_IL7-AS-S2-.jpg
 
  
 
===References===
 
===References===
 
Roux, B. T., Heward, J. A., Donnelly, L. E., Jones, S. W., and Lindsay, M. A. (2017) Catalog of Differentially Expressed Long Non-Coding RNA following Activation of Human and Mouse Innate Immune Response. Frontiers in immunology 8, 1038
 
Roux, B. T., Heward, J. A., Donnelly, L. E., Jones, S. W., and Lindsay, M. A. (2017) Catalog of Differentially Expressed Long Non-Coding RNA following Activation of Human and Mouse Innate Immune Response. Frontiers in immunology 8, 1038

Latest revision as of 07:44, 11 October 2018


pSB1C3-IL7-AS-S2


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 198
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We wanted to investigate which domain is essential for the function of IL7-AS. We cloned the truncated sequences of IL7-AS (IL7-AS-S2) into pSB1C3 for submitting to IGEM 2018 and inserted IL7-AS-S2 into pCDNA3.1 to study the function.


Source

We cloned the truncated sequences of IL7-AS (IL7-AS-S2,411 bp) from human cells.

1.1 Amplification of IL7-AS-S2 fragments from human cell line. First we amplified IL7-AS-S2 using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with restriction enzymes EcoRI and PstI (Fig 1).

Amplification_of_IL7-AS_and_IL7-AS-S2.jpeg

1.2 Digested PSB1C3 vector. We digested the PSB1C3 vectors with EcoRI and PstI (Fig 2).

Digested_the_PSB1C3_vectors_with_EcoRI_and_PstI.jpeg


1.3 Ligation of purified IL7-AS-S2 fragments to pSB1C3 vector.


IL7-AS-S2 fragments were ligated to PSB1C3 vector. Then we selected the positive clones by PCR and sequencing (Fig 3). Verify_pSB1C3-IL7-AS-S2_by_sequencing.jpeg

References

Roux, B. T., Heward, J. A., Donnelly, L. E., Jones, S. W., and Lindsay, M. A. (2017) Catalog of Differentially Expressed Long Non-Coding RNA following Activation of Human and Mouse Innate Immune Response. Frontiers in immunology 8, 1038