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

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pSB1C3-IL7-AS-S2

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).

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

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).

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, IL7-AS-S1 and 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).

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.

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)

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