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

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We cloned the truncated sequences of IL7-AS (IL7-AS-S2,411 bp) from human cells.
 
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.
 
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).
 
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).
 +
 
https://static.igem.org/mediawiki/parts/e/e0/Amplification_of_IL7-AS_and_IL7-AS-S2.jpeg
 
https://static.igem.org/mediawiki/parts/e/e0/Amplification_of_IL7-AS_and_IL7-AS-S2.jpeg
 +
 
1.2 Digested PSB1C3 vector.
 
1.2 Digested PSB1C3 vector.
 
We digested the PSB1C3 vectors with  EcoRI and PstI (Fig 2).
 
We digested the PSB1C3 vectors with  EcoRI and PstI (Fig 2).
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https://parts.igem.org/File:Digested_the_PSB1C3_vectors_with_EcoRI_and_PstI.jpeg
+
 
 +
https://static.igem.org/mediawiki/parts/5/5b/Digested_the_PSB1C3_vectors_with_EcoRI_and_PstI.jpeg
 +
 
 +
 
 
1.3 Ligation of purified IL7-AS-S2 fragments to pSB1C3 vector.
 
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).
 
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

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