Team:Worldshaper-Wuhan/Basic Part

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pSB1C3-IL7-AS-S2




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IL7-AS [located anti-sense to interleukin-7 (Il7) gene; Accession number: NM_000880.3] is a newly discovered lncRNA that can be induced across multiple human and mouse cell types and has been reported to regulate the expression of interleukin-6 (IL6). IL6 is emerging as a major regulator of renal cell cancer. However, roles of IL7-AS in renal cell cancer and the related molecular mechanisms remain largely undetermined.We cloned the truncated sequences of IL7-AS (IL7-AS-S2 contain 411bp) into pSB1C3 for submitting. And we also cloned IL7-AS-S2 into PCDNA3.1 and transfected these plasmids into 786-O cells. Through in vitro scratch wound healing assay, overexpression of IL7-AS-S2 promoted cell migration of 786-O cells. These results suggested that IL7-AS-S2 contain key structural domains.

Plates from our first try

(Fig. Scheme of three IL7-AS truncated sequences on human chromosome 8)




Construction Of The Plasmids

We constructed three plasmids PSB1C3-IL7-AS-S2; PSB1C3-IL7-AS-S1 and PSB1C3-IL7-AS. And these three plasmids were submitted to 2018IGEM.

  1. Amplification of IL7-AS-S2; IL7-AS-S1 and IL7-AS fragments. First we amplified IL7-AS-S2; IL7-AS-S1 and IL7-AS fragment using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with restriction enzymes EcoRI and PstI (Fig 1 A and B).
    2. Fig.1A and Fig.1B

    (Fig 1. Electrophoresis of IL7-AS-S2 (A: lanes 1); IL7-AS (A: lanes 2) and IL7-AS-S1 (B: lanes3) PCR product.)



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

    (Fig 2. Electrophoresis of the digested PSB1C3 vector.)



  3. Ligation of purified IL7-AS-S2; IL7-AS-S1 and IL7-AS fragments to PSB1C3 vector. IL7-AS-S2(A); IL7-AS-S1(B) and IL7-AS(C) fragments were ligated to PSB1C3 vector, respectively. Then we selected the positive clones by PCR and sequencing (Fig 3).
    4. Fig.3a

    (Fig 3.A)



    Fig.3b

    (Fig 3.B)



    Fig.3c

    (Fig 3.C)



Ligation of purified IL7-AS-S2 fragments to pCDNA3.1 vector



Fig.4



The secondary structures of IL7-AS-S2 were predicted as below



Fig.5