Team:UT-Tokyo/Project/Inhibition/Discussion

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{{:Team:UT-Tokyo/Template/Header|fullpagename=|subpagename=Inhibition without Knockout: <br />Results & Discussion}}
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このページの概要を、簡単に記述。
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==LacI - pLac==
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LacI represses the frequently used promoter, pLac. The binding of LacI to pLac is competitively inhibited and the repression of pLac is weakened by introducing 8 LacI-binding-sites on pSB1C3 (a high-copy-plasmid).
 +
By introducing 8 tandem repeats of LacI binding sites on pSB1C3 in addition to pLac-RBS-GFP-d.term on pSB1A2, it is expected that the binding of LacI to pLac is competitively inhibited which can be visually tracked by GFP expression. Moreover, longer LacI binding sites  (more than 8 binding sites) is thought to capture more LacI and enhance GFP expression more effectively.
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== 編集の仕方 ==
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==ArgR - ArgR binding site==
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* ページ右上にあるログインリンクからログインできます。
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rocF is the gene coding the enzyme that converts L-arginine to L-ornithine and urea. By introducing rocF, e.coli obtains the urea cycle. ArgR is the common repressor of the bacterial arginine biosynthetic genes. By introducing ArgR binding sites, the probability that ArgR binds to the operator of the arginine biosynthetic genes falls and arginine biosynthesis is derepressed. (Tokyo Tech 2011)
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* ログイン済みの場合は、ページ左上にカーソルを持っていけば、editから内容を編集できます。(日本語メニューの場合は「編集」)
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By introducing ArgR binding sites (including 8 binding sites) on pSB1C3 (high-copy-plasmid) in addition to Ptrc-RBS-rocF on pSB6A1 (low-copy-plasmid), ArgR is sequestered by ArgR binding sites and arginine biosynthesis is derepressed, so that urea production rate is expected to rise compared to Ptrc-RBS-rocF on low-copy-plasmid / ArgR binding site on high-copy-plasmid (Tokyo Tech 2011). Furthermore, although a single ArgR binding site introduced downstream of Ptrc-RBS-rocF did not derepress arginine biosynthesis, it is thought that sufficiently long ArgR binding sites introduced downstream the Ptrc-RBS-rocF binds to many more ArgR proteins and derepresses arginine biosynthesis, consequently enhancing the urea production rate.
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* 新規ページを作るには、アドレスバーに作りたいページのURLを打ち込めばできます。そのページには、このテンプレートページの内容を全てコピーして貼り付け、指定がある部分を編集して自由記述すればOKです。
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== wikiの記法 ==
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[[File:UT Tokyo Fig knockdown-hycAp 640 240.png|480]]
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[http://2008.igem.org/Team:Chiba/Internal/foredit 2008年度Team:Chibaのwiki]が参考になります。
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== Modeling ==
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そのページでもリンクされていますが、書き方は
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[http://ja.wikipedia.org/wiki/Help:%E3%83%9A%E3%83%BC%E3%82%B8%E3%81%AE%E7%B7%A8%E9%9B%86 Wikipedia-Help:ページの編集]準拠のようです。
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* 表の書き方:[http://ja.wikipedia.org/wiki/Help:%E8%A1%A8%E3%81%AE%E4%BD%9C%E3%82%8A%E6%96%B9 wikipedia Help:表の作り方]
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To assess the effectiveness of our method, we made a model and to calculate estimated differences in transcription factor availability with and without adding tandem repeats of binding sites.
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== 見出し1 ==
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We start with up the equilibrium reaction:<br />
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*[[File:UT Tokyo-TF-DNA.png|Alt="Transcription Factor + DNA ←→ Transcription Factor-DNA"|x18px]]
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== 見出し2 ==
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By introducing binding sites into high-copy plasmids, the following reactions are added:<br />
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=== 小見出し1 ===
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*[[File:UT Tokyo TF-Genome.png|Alt="Transcription Factor+ Genome DNA ←→ Transcription Factor-Genome DNA"|x18px]] <br />
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*[[File:UT Tokyo TF-bs.png|Alt="Transcription Factor + Binding Site on Plasmid ←→Transcription Factor-Binding Site"|x18px]]
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リンクの例:<br />
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Let
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*ページ内リンク
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*[I]:=concentration of the transcription factor alone
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**右のように[[#見出し1]]と書くと見出し1へのリンクが張られます。ページトップ[[#top]]と、各見出しへはこのようにしてリンクが張れます。
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*[G]:=concentration of the genomic DNA alone
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*Wiki内部リンク
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*[GI]:=concentration of the  transcription factor - genome DNA complex
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**右のように[[Team:UT-Tokyo/Internal/Sandbox]] と書くとそのまま表示され<br />
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*[T]:=concentration of the binding site on plasmid alone
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**右のように[[Team:UT-Tokyo/Internal/Sandbox|Sandboxへのリンク]]と書くと「Sandboxへのリンク」という文字列にリンクが張られます。
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*[TI]:concentration of the transcriptional factor - binding site complex
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*Wiki外部リンク
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Also, we assumed that the equilibrium constants, k, are equal.
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**右のようにhttp://2012.igem.org/Team:UT-Tokyoと書くとそのまま表示され、
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[[File:UT Tokyo k.png|150px|Alt="k:=[GI]/[I][G]=[TI]/[I][T]"]]
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**右のように[http://2012.igem.org/Team:UT-Tokyo UT-Tokyoのトップページ]と書くと「UT-Tokyoのトップページ」という文字列にリンクが張られます。
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**右のように[http://2012.igem.org/Team:UT-Tokyo]と書くと、自動で番号のついたリンクが張られます。
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画像にリンクしたい場合:
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This leads to
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[[media:example.jpg]]
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*[GI] = k [I][G]
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*[TI] = k [I][T]
 +
*[I]+[GI]+[TI]=const=:X (Total concentration of the transcription factor)
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*[G]+[GI]=const=:Y (Total concentration of the genomic DNA)
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*[T]+[TI]=const=:Z (Total concentration of the binding site on plasmid)
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画像を表示したい場合
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What we want to know is how [[File:UT Tokyo GI-Y.png|30px|Alt="[GI]/Y"]] changes from a situation where [G]:[T] is 1:0 (wild type) to 1:1000 (introducing 10 tandem binding sites into a 100 copy  plasmid), because [[File:UT Tokyo GI-Y.png|30px|Alt="[GI]/Y"]] represents the proportion of Genomic DNA bound by the transcription factor. Let u:= [[File:UT Tokyo GI-Y.png|30px|Alt="[GI]/Y"]].
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[[File:ファイル名(拡張子込)]]
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 +
By substitution,
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[[File:UT Tokyo -GI- Y.png|400px]]
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段落内改行は<br />
 
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;定義リストの定義
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we set the variables y:=Y/X, z:=Z/X, t:=1/kX.
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:定義リストの説明
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== 画像類 ==
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=== For LacI ===
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[[Team:UT-Tokyo/Internal/Images]]にがぞうのせてる
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Now, we solve this equation for the LacI transcription factor because its k is known.
 +
 
 +
the equilibrium constant [[File:UT Tokyo k LacI.png|300px]]
 +
for the equilibrium [1]<br />
 +
*[[File:UT Tokyo-LacIDNA.png|Alt="LacI + DNA ←→ LacI-DNA"|300px]]
 +
 
 +
 
 +
The concentration of genomic DNA is [[File:UT Tokyo GenomeConc.png|300px|Alt="0.88x10^(-15)/6.02*10^23 = 1.887*10^(-9) [l/mol]"]][2]
 +
 
 +
 
 +
From the above, we caluculated u against X (Total concentration of the transcription factor) for several values of Z/Y(the number of binding sites introduced) = 0, 1, 10, 100, 1000 and obtained the graph following
 +
 
 +
 
 +
[[File:UT_Tokyo_IG-graph8.jpg]]
 +
 
 +
 
 +
 
 +
This graph shows the obvious effectiveness of introducing LacI binding sites into a high-copy plasmid (when the copy number ~ 10^2). Also, it shows that If X is high the effect of introducing tandem repeat is greater(by comparing Z/Y = 100 and 1000).
 +
 
 +
=== With Larger/Smaller k Than That of LacI ===
 +
 
 +
For reference, we write some graphs with larger/smaller k, to apply to other transcription factors.
 +
 
 +
 
 +
If k==10^6:
 +
 
 +
[[File:UT_Tokyo_IG-graph6.jpg]]
 +
 
 +
 
 +
If k==10^7:
 +
 
 +
[[File:UT_Tokyo_IG-graph7.jpg]]
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 +
 
 +
If k==2.4 * 10^8 (LacI)
 +
 
 +
[[File:UT_Tokyo_IG-graph8.jpg]]
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 +
 
 +
If k==10^10:
 +
 
 +
[[File:UT_Tokyo_IG-graph10.jpg]]
 +
 
 +
 
 +
These graphs show that as k is increased, the effect of introducing binding sites becomes bigger.
 +
 
 +
 
 +
=== Reference ===
 +
*[1] LactoseRepressor-OperatorDNA Interactions: KineticAnalysis by aSurfacePlasmonResonanceBiosensor K. Bondeson, A. Frostellkarlsson, L. Fagerstam, G. Magnusson Univ Uppsala, Ctr Biomed, Dept Med Virol, S 75123 Uppsala, Sweden and Pharmacia Biosensor AB, S 75182 Uppsala, Sweden
 +
*[2] CCDB ''E.coli'' stastics http://ccdb.wishartlab.com/CCDB/cgi-bin/STAT_NEW.cgi
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;[[#見出し1|見出し1or子下階層ページ名1]]
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;[[#LacI - pLac|LacI - pLac]]
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:題名1の説明<br />改行して説明の続き
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;[[#見出し2|見出し2or子階層ページ名2]]
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;[[#ArgR - ArgR binding site|ArgR - ArgR binding site]]
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:説明
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;見出し2or子階層ページ名3
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;[[#Modeling|Modeling]]
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:description
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*[[#For LacI|For LacI]]
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;見出し2or子階層ページ名4
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*[[#With Larger/Smaller k Than That of LacI|With Larger/Smaller k Than That of LacI]]
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:description
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*[[#Reference|Reference]]
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{{:Team:UT-Tokyo/Template/Footer|prevname=Inhibition Without Knockout: Background & System|prevfull=Project/Inhibition/System|nextname=Parts|nextfull=Parts}}

Latest revision as of 03:09, 27 September 2012

Inhibition without Knockout:
Results & Discussion

box-background image

LacI - pLac

LacI represses the frequently used promoter, pLac. The binding of LacI to pLac is competitively inhibited and the repression of pLac is weakened by introducing 8 LacI-binding-sites on pSB1C3 (a high-copy-plasmid). By introducing 8 tandem repeats of LacI binding sites on pSB1C3 in addition to pLac-RBS-GFP-d.term on pSB1A2, it is expected that the binding of LacI to pLac is competitively inhibited which can be visually tracked by GFP expression. Moreover, longer LacI binding sites (more than 8 binding sites) is thought to capture more LacI and enhance GFP expression more effectively.

ArgR - ArgR binding site

rocF is the gene coding the enzyme that converts L-arginine to L-ornithine and urea. By introducing rocF, e.coli obtains the urea cycle. ArgR is the common repressor of the bacterial arginine biosynthetic genes. By introducing ArgR binding sites, the probability that ArgR binds to the operator of the arginine biosynthetic genes falls and arginine biosynthesis is derepressed. (Tokyo Tech 2011) By introducing ArgR binding sites (including 8 binding sites) on pSB1C3 (high-copy-plasmid) in addition to Ptrc-RBS-rocF on pSB6A1 (low-copy-plasmid), ArgR is sequestered by ArgR binding sites and arginine biosynthesis is derepressed, so that urea production rate is expected to rise compared to Ptrc-RBS-rocF on low-copy-plasmid / ArgR binding site on high-copy-plasmid (Tokyo Tech 2011). Furthermore, although a single ArgR binding site introduced downstream of Ptrc-RBS-rocF did not derepress arginine biosynthesis, it is thought that sufficiently long ArgR binding sites introduced downstream the Ptrc-RBS-rocF binds to many more ArgR proteins and derepresses arginine biosynthesis, consequently enhancing the urea production rate.

480

Modeling

To assess the effectiveness of our method, we made a model and to calculate estimated differences in transcription factor availability with and without adding tandem repeats of binding sites.

We start with up the equilibrium reaction:

  • Alt="Transcription Factor + DNA ←→ Transcription Factor-DNA"

By introducing binding sites into high-copy plasmids, the following reactions are added:

  • Alt="Transcription Factor+ Genome DNA ←→ Transcription Factor-Genome DNA"
  • Alt="Transcription Factor + Binding Site on Plasmid ←→Transcription Factor-Binding Site"

Let

  • [I]:=concentration of the transcription factor alone
  • [G]:=concentration of the genomic DNA alone
  • [GI]:=concentration of the transcription factor - genome DNA complex
  • [T]:=concentration of the binding site on plasmid alone
  • [TI]:concentration of the transcriptional factor - binding site complex

Also, we assumed that the equilibrium constants, k, are equal. Alt="k:=[GI]/[I][G]=[TI]/[I][T]"

This leads to

  • [GI] = k [I][G]
  • [TI] = k [I][T]
  • [I]+[GI]+[TI]=const=:X (Total concentration of the transcription factor)
  • [G]+[GI]=const=:Y (Total concentration of the genomic DNA)
  • [T]+[TI]=const=:Z (Total concentration of the binding site on plasmid)

What we want to know is how Alt="[GI]/Y" changes from a situation where [G]:[T] is 1:0 (wild type) to 1:1000 (introducing 10 tandem binding sites into a 100 copy plasmid), because Alt="[GI]/Y" represents the proportion of Genomic DNA bound by the transcription factor. Let u:= Alt="[GI]/Y".

By substitution, UT Tokyo -GI- Y.png


we set the variables y:=Y/X, z:=Z/X, t:=1/kX.

For LacI

Now, we solve this equation for the LacI transcription factor because its k is known.

the equilibrium constant UT Tokyo k LacI.png for the equilibrium [1]

  • Alt="LacI + DNA ←→ LacI-DNA"


The concentration of genomic DNA is Alt="0.88x10^(-15)/6.02*10^23 = 1.887*10^(-9) [l/mol]"[2]


From the above, we caluculated u against X (Total concentration of the transcription factor) for several values of Z/Y(the number of binding sites introduced) = 0, 1, 10, 100, 1000 and obtained the graph following


UT Tokyo IG-graph8.jpg


This graph shows the obvious effectiveness of introducing LacI binding sites into a high-copy plasmid (when the copy number ~ 10^2). Also, it shows that If X is high the effect of introducing tandem repeat is greater(by comparing Z/Y = 100 and 1000).

With Larger/Smaller k Than That of LacI

For reference, we write some graphs with larger/smaller k, to apply to other transcription factors.


If k==10^6:

UT Tokyo IG-graph6.jpg


If k==10^7:

UT Tokyo IG-graph7.jpg


If k==2.4 * 10^8 (LacI)

UT Tokyo IG-graph8.jpg


If k==10^10:

UT Tokyo IG-graph10.jpg


These graphs show that as k is increased, the effect of introducing binding sites becomes bigger.


Reference

  • [1] LactoseRepressor-OperatorDNA Interactions: KineticAnalysis by aSurfacePlasmonResonanceBiosensor K. Bondeson, A. Frostellkarlsson, L. Fagerstam, G. Magnusson Univ Uppsala, Ctr Biomed, Dept Med Virol, S 75123 Uppsala, Sweden and Pharmacia Biosensor AB, S 75182 Uppsala, Sweden
  • [2] CCDB E.coli stastics http://ccdb.wishartlab.com/CCDB/cgi-bin/STAT_NEW.cgi