RNA

Part:BBa_K2889000:Design

Designed by: Rui Zhou   Group: iGEM18_Worldshaper-Wuhan   (2018-10-04)
Revision as of 07:16, 11 October 2018 by Ruizhou (Talk | contribs) (Source)


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


In silico prediction of lncRNA secondary structure is another useful method to assign putative functions to non-coding transcripts, based upon the widely held assumption that highly folded structures impart functionality through binding interactions with proteins/nucleotides. Characterization of IL7-AS-S2 using RNAfold minimum free energy estimations predicted a highly folded secondary structure with several hairpin loops.The secondary structure of IL7-AS-S2(Fig 4).


IL7-AS-S2.jpg


The optimal secondary structure with a minimum free energy The secondary structures of IL7-AS; IL7-AS-S1 and IL7-AS-S2 were predicted by the RNAfold webserver (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). The structure is colored according to base-pairing probabilities. For unpaired regions, the color denotes the probability of being unpaired.


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.


Scheme_of_three_IL7-AS_isoforms.jpg


IL7-AS-AS2.jpg


IL7-AS-S2-cell_migration.jpg


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)


Different_concentration_of_IL7-AS-S2.jpg


Different_concentration_of_IL7-AS-S2-.jpg

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