iGEM 2012 — Team Nanjing-China

[![](/wiki/skins/common/images/wiki.png)"](/Main_Page "Main Page")

* [Page](/Team%3ANanjing-China/Notebook)
* [Discussion](/wiki/index.php?title=Talk:Team:Nanjing-China/Notebook&action=edit&redlink=1)
* [View source](/wiki/index.php?title=Team:Nanjing-China/Notebook&action=edit)
* [History](/wiki/index.php?title=Team:Nanjing-China/Notebook&action=history)
* teams

* [Log in](/wiki/index.php?title=Special:UserLogin&returnto=Team:Nanjing-China/Notebook "You are encouraged to log in; however, it is not mandatory [o]")

# Team:Nanjing-China/Notebook

### From 2012.igem.org

* [Home](https://2012.igem.org/Team%3ANanjing-China)
* [Team](https://2012.igem.org/Team%3ANanjing-China/Team)
* [Profile](https://igem.org/Team.cgi?year=2012&team_name=Nanjing-China)
* [Project](https://2012.igem.org/Team%3ANanjing-China/Project)
* [Parts](https://2012.igem.org/Team%3ANanjing-China/Parts)
* [Partner](https://2012.igem.org/Team%3ANanjing-China/Partner)
* [Notebook](https://2012.igem.org/Team%3ANanjing-China/Notebook)
* [Safety](https://2012.igem.org/Team%3ANanjing-China/Safety)
* [Attributions](https://2012.igem.org/Team%3ANanjing-China/Attributions)
* [Practise](https://2012.igem.org/Team%3ANanjing-China/Practise)

# The steps of the experiments

## 1. Design of amiRNA Sequences

The target gene we want to silence is PGC-1a which is related to metabolism in several tissues. After inputting the sequence of PGC-1a to the Web MicroRNA Designer, the software will give us several potential amiRNA sequences which have high possibilities to silence the target genes. Although the sequences are picked out based on several principles, we still need to select the sequences by ourselves in the rules:

* It is preferable for all intended target genes to not have mismatches to the amiRNA at positions 2 to 12.
* AmiRNA candidates with one or two mismatches at the 3¢ end of the amiRNA (positions 18 to 21) should be preferred, since it has been suggested that perfectly matching amiRNAs might trigger so-called transitive siRNA formation, where amplification of sequences adjacent to the binding site is primed by the miRNA. These sequences could in turn themselves serve as silencing triggers and affect other, unintended genes.
* The absolute hybridization energy of the binding between amiRNA and the target sequence should be less than −30 kcal/ mole, and preferable be in the range between −35 and −40 kcal/ mole.
* The amiRNA binding site should be located within the coding region of the target gene, since UTRs are more likely to be misannotated. At least two amiRNAs per target gene or group of genes should be selected for experimental work. If several are selected, the amiRNAs should bind the target mRNA at different locations, since secondary structure is suspected to influence miRNA efficacy.

Of course, there is still an important thing to do before we use the sequences to do experiments. That is we should to do BLAST research to check whether the sequences have binding sites in other genes which will greatly affect the experiment.

After all these work we can get the amiRNA sequences which may work well during the experiments.

## 2. Construction of aMIRNA precursors by Site-Directed Mutagenesis

What we want to do is to make a miRNA precursor and make use of the Dicer in the plants to produce amiRNA which we design in the former step. MIRNA precursors fold back on themselves to form a hairpin structure, and it is important to preserve this structure for successful processing. Therefore, engineering of amiRNAs into MIRNA precursor templates not only requires the exchange of the miRNA by the amiRNA sequence, but also of the pairing region in the hairpin, called the (a)miRNA\*, such that pairing positions as well as G:U pairs are retained. The WMD software (WMD-Oligo window) thus generates four oligonucleotides per amiRNA sequence input: I and II to engineer the actual amiRNA, and III and IV for the amiRNA\* (with wobbles).

![](https://static.igem.org/mediawiki/2012/a/a8/Notebook-a.png)

[![Powered by MediaWiki](/wiki/skins/common/images/poweredby_mediawiki_88x31.png)](http://www.mediawiki.org/)

[![Attribution 3.0 Unported](http://i.creativecommons.org/l/by/3.0/88x31.png)](http://creativecommons.org/licenses/by/3.0/)

* [Recent changes](/Special%3ARecentChanges "Recent changes")
* [What links here](/Special%3AWhatLinksHere/Team%3AHokkaidoU_Japan "List of all wiki pages that link here [j]")
* [Related changes](/Special%3ARecentChangesLinked/Team%3AHokkaidoU_Japan "Recent changes in pages linked from this page [k]")
* [Upload file](/Special%3AUpload "Upload files [u]")
* [Special pages](/Special%3ASpecialPages "List of all special pages [q]")
* [My preferences](/Special%3APreferences)

* [Printable version](/wiki/index.php?title=Team:HokkaidoU_Japan&printable=yes "Printable version of this page [p]")
* [Permanent link](/wiki/index.php?title=Team:HokkaidoU_Japan&oldid=263009 "Permanent link to this revision of the page")
* [Privacy policy](/2011.igem.org%3APrivacy_policy "2011.igem.org:Privacy policy")
* [Disclaimers](/2011.igem.org%3AGeneral_disclaimer "2011.igem.org:General disclaimer")

* [![](https://static.igem.org/mediawiki/igem.org/4/46/Nju_logo.png)](www.nju.edu.cn)
* [![](https://static.igem.org/mediawiki/igem.org/0/08/Igem_logo.png)](igem.org/Main_Page)

Retrieved from "[http://2012.igem.org/Team:Nanjing-China/Notebook](http://2012.igem.org/Team%3ANanjing-China/Notebook)"

[![Powered by MediaWiki](/wiki/skins/common/images/poweredby_mediawiki_88x31.png)](https://www.mediawiki.org/)

[![Attribution 3.0 Unported](https://i.creativecommons.org/l/by/3.0/88x31.png)](https://creativecommons.org/licenses/by/3.0/)

* [Recent changes](/Special%3ARecentChanges "Recent changes")
* [What links here](/Special%3AWhatLinksHere/Team%3ANanjing-China/Notebook "List of all wiki pages that link here [j]")
* [Related changes](/Special%3ARecentChangesLinked/Team%3ANanjing-China/Notebook "Recent changes in pages linked from this page [k]")
* [Special pages](/Special%3ASpecialPages "List of all special pages [q]")
* [My preferences](/Special%3APreferences)

* [Printable version](/wiki/index.php?title=Team:Nanjing-China/Notebook&printable=yes "Printable version of this page [p]")
* [Permanent link](/wiki/index.php?title=Team:Nanjing-China/Notebook&oldid=234646 "Permanent link to this revision of the page")
* [Privacy policy](/2012.igem.org%3APrivacy_policy "2012.igem.org:Privacy policy")
* [Disclaimers](/2012.igem.org%3AGeneral_disclaimer "2012.igem.org:General disclaimer")