Team:UT-Tokyo/Sudoku lab note

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= '''Sudoku''' =
= '''Sudoku''' =
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[https://2010.igem.org/Team:UT-Tokyo/Sudoku_abstract  Abstract]     
+
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_abstract  Introduction]     
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[https://2010.igem.org/Team:UT-Tokyo/Sudoku_construct  Construct]    
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[https://2010.igem.org/Team:UT-Tokyo/Sudoku_construct  System]
 +
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_modeling  Modeling]       
Lab note       
Lab note       
[
[
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_notebook_6  June]    /
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_notebook_6  June]    /
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_notebook  July]    /
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_notebook  July]    /
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[https://2010.igem.org/Team:UT-Tokyo/Sudoku_notebook_2  August]     
+
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_notebook_2  August]    / 
 +
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_notebook_8  September - October]
]
]
 +
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_experiments  Experiment]
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_result  Result]
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_result  Result]
 +
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_reference  Reference]
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== '''Lab note''' ==
 
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To make main construct, we made several assays.
 
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1. [[#Terminator leak switch]]
 
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2. [[#Location sequence]]
 
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3. [[#MS2 virus]]
 
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4. [[#flpe]]
 
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5. [[#Hin]]
 
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Detail protocols about this is:
 
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[https://2010.igem.org/Team:UT-Tokyo/Sudoku_notebook_6  June].
 
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[https://2010.igem.org/Team:UT-Tokyo/Sudoku_notebook July].
 
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[https://2010.igem.org/Team:UT-Tokyo/Sudoku_notebook_2 August].
 
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= Main construct =
 
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[[Image:Sudoku_const_Main_5.png|200px|thumb|Main construct]]
 
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If you want to know detail explanation, please read [https://2010.igem.org/Team:UT-Tokyo/Sudoku_construct  Construct] section.
 
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= Assay =
 
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== Terminator leak switch ==
 
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[[Image:Sudoku_Assay_terminator_3.png|200px|thumb|Terminator leak switch assay]]
 
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To realize 4C3 leak switch, we should find a proper terminator which terminates transcription when connected two or more but leaks when single.
 
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We made "A-M-Ro/Char" assay (see Fig, named from the famous animation, "MOBILE SUIT GUNDAM") to select proper terminator:
 
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'''Aoi'''
 
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no terminator
 
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-> cre protein express rapidly
 
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-> lox site is removed rapidly
 
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-> gfp may expression rapidly
 
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'''Murasaki'''
 
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one terminator
 
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-> cre protein express slowly
 
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-> lox site is removed slowly
 
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-> gfp may expression slowly
 
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'''Rokujyo'''
 
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two terminator
 
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-> cre protein can't express
 
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-> lox site remain
 
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-> gfp expression may not express
 
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First we use single terminator BBa_B1006.
 
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Then other terminators are tested: 80%-terminate terminator and 99%-terminate terminator to determine the best threshold to realize terminator leak switch.
 
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== Location sequence ==
 
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===Parts making===
 
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[[Image:Sudoku_location_making.png|200px|thumb|How to make location parts]]
 
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Ligate small parts - recognation site, location site and rbs.
 
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===Expression check===
 
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[[Image:Sudoku_Assay_location_4.png|200px|thumb|Location sequence]]
 
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This assay testifies whether translation repression by location sequence go well.
 
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First we check the ability of pBAD, which is induced by arabinose, by the expression of tetracycline-tolerance protein.
 
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Then to check translation repression, we make assay which express gfp only when translation repression can’t be occurring.
 
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== MS2 virus ==
 
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===Parts making===
 
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[[Image:Sudoku_MS2-make_2.jpg|200px|thumb|How to make MS2 parts]]
 
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We get MS2 gene RT-PCR product.
 
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This original product include a lot of restricted enzyme site:
 
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two EcoRI site, two XbaI site and one VspI site.
 
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To run our project, we don’t have to remove EcoRI site in the region.
 
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So we modified XbaI site by PCR as the following method:
 
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1. We divided RT-PCR product into two parts by XP enzyme digestion:
 
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E-X-E-E-V-X -> “Goten”
 
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X-S-P -> “Tranks”
 
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(named from the famous animation, "DRAGON BALL")
 
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2. Insert is ligated with the vector, chloramphenicol-tolerance:
 
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Goten -> EX vector
 
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Tranks -> XP vector
 
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3. Tranks -> PCR adding V-X region
 
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4. Each part -> VP enzyme digestion, ligation each other
 
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===Expression check===
 
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[[Image:Sudoku_Assay_MS2_5.png|200px|thumb|Virus expression check assay]]
 
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We use MS2 phage to transmit information of location and number.
 
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The expression of the phage should start after 4C3 leak switch turns on.
 
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MS2 phage transport RNA which has loding sequence, so we knock out self assembly and made our E.coli translate loading seaquence at another point.
 
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To check whether transportion go correctly, we made assay.
 
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i. Phage expression assay (red region)
 
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Check whether RT-PCR product can be translated into MS2 phage.
 
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ii. Packaging assay (blue & green region)
 
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MS2 phage package RNA which has loading sequence. Using this character, we check whether RNA which has loading sequence and other coding region (gfp, cre) can be packaged correctly.
 
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iii. Infection assay (yellow & green region)
 
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We make the assay, which express gfp only when cre protein is expressed correctly.
 
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By using this part, we check whether E.coli can be infected with the MS2 phage made in assay ii as the fluorescence of gfp.
 
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== flpe ==
 
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===Parts making===
 
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[[Image:Sudoku_flpe_making.png|200px|thumb|How to make flpe parts]]
 
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The original flpe include two restriction enzyme sites(EcoRI, SpeI).
 
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We modified this flpe by PCR:
 
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1st PCR : cloning
 
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-> ligation with vector
 
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-> 2nd PCR : modified SpeI site
 
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-> 3rd PCR : modified EcoRI site
 
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We use this part as the form of reverse, so we made this part reverse by PCR.
 
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===Expression check===
 
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[[Image:Sudoku_Assay_flpe_2.png|200px|thumb|flpe expression check assay]]
 
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To check whether flpe works correctly, we made assay shown in Fig.
 
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The top construct is a nagative control.
 
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GFP can't be expressed because of the double terminators.
 
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The bottom construct express GFP when flpe works correctly.
 
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When flpe is expressed correctly, flpe recognaize the frt site and double terminator which terminates the expression of gfp is removed, so GFP may be expressed.
 
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== Hin ==
 
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===Parts making (reverse)===
 
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[[Image:Sudoku_Hin_making.png|200px|thumb|How to make Hin parts]]
 
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We get Hin parts from HQ (BBa_J31000).
 
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We use this part as the form of reverse, so we made this part reverse by PCR.
 
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To check PCR is done correctly, we did VspI digestion.
 
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===Expression check===
 
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[[Image:Sudoku_Assay_Hin.png|200px|thumb|Hin expression check assay]]
 
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To check whether flpe works correctly, we made assay shown in Fig, similar to the assay of flpe check.
 
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The top construct is a nagative control.
 
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GFP can't be expressed because of the double terminators.
 
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The bottom construct express GFP when Hin works correctly.
 
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When Hin is expressed correctly, Hin recognaize the hix site and double terminator which terminates the expression of gfp is removed, so GFP may be expressed.
 
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= Parts list =
 
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{| border="1"
 
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| Number || Name || Link to BioBrick || Plate coordinate || Vector || Code length
 
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|-
 
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| 1 || T7 promoter ||
 
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_I712074  BBa_I712074]
 
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|| plate1-6N || pSB1AK8 || 46bp
 
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|-
 
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| 2 || rbs ||
 
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0030  BBa_B0030]
 
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|| plate1-1H || pSB1A2 || 15bp
 
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|-
 
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| 3 || cre recombinase ||
 
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_J61047  BBa_J61047]
 
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|| plate1-5D || pSB1A2 || 1037bp
 
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|-
 
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| 4 || double terminator ||
 
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0014  BBa_B0014]
 
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|| plate2-24C || pSB1AK3 || 95bp
 
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|-
 
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| 5 || lox66 recombinase site ||
 
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_I718017  BBa_I718017]
 
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|| plate1-17J || pSB1A2 || 34bp
 
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|-
 
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| 6 || Kan resistance (rev) ||
 
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_J31002  BBa_J31002]
 
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|| plate1-2K || pSB1A2 || 816bp
 
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|-
 
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| 7 || rbs (rev) ||
 
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0014  BBa_B0014]
 
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|| plate1-1J || pSB1A2 || 15bp
 
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|-
 
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| 8 || lox71 recombinase site ||
 
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order-made
 
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|| --- || --- || 34bp
 
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|-
 
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| 9 || single terminator ||
 
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_B1006  BBa_B1006]
 
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|| plate1-4H || pSB1AK3 || 39bp
 
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|-
 
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| 10 || constant express promoter ||
 
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23119  BBa_J23119]
 
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|| plate1-18A || pSB1A2 || 35bp
 
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|-
 
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| 11 || Tet resistance (rev) ||
 
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_J31006  BBa_J31006]
 
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|| plate1-1N || pSB1A2 || 1191bp
 
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|-
 
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| 12 || hixC ||
 
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_J44000  BBa_J44000]
 
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|| plate1-1B || pSB1A2 || 26bp
 
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|-
 
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| 13 || --- ||
 
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---
 
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|| --- || --- || ---
 
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|-
 
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| 14 || lox66 ||
 
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_I718016  BBa_I718016]
 
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|| plate1-17H || pSB1A2 || 34bp
 
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|-
 
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| 15 || rbs-mRFP1-terminator ||
 
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_I13507  BBa_I13507]
 
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|| plate1-22O || pSB1A2 || 861bp
 
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|-
 
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| 16 || location sequence(old) ||
 
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order
 
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|| --- || --- || ---
 
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|-
 
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| 17 || pSP6(rev) ||
 
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order
 
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|| --- || --- || ---
 
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|-
 
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| 18 || lox2272 ||
 
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order
 
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|| --- || --- || ---
 
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|-
 
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| 19 || loading sequence(rev) ||
 
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order
 
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|| --- || --- || ---
 
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|-
 
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| 20 || frt ||
 
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[http://partsregistry.org/Part:BBa_J61020  BBa_J61020]
 
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|| From HQ || pSB1A2 || 34bp
 
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|-
 
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| 21 || hin(true) ||
 
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[http://partsregistry.org/Part:BBa_J31000  BBa_J31000]
 
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|| From HQ || ??? || 573bp
 
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|-
 
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| 22 || hin(rev) ||
 
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---
 
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|| --- || ??? || 573bp
 
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|-
 
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| 23 || flpe ||
 
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---
 
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|| --- || --- || about 1.2kbp
 
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|-
 
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| 24 || gfp unit ||
 
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---
 
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|| --- || --- || about 1.2kbp
 
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|-
 
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| 25 || flpe(rev) ||
 
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---
 
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|| --- || --- || about 1.2kbp
 
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|}
 
{{UT-Tokyo_Foot}}
{{UT-Tokyo_Foot}}

Latest revision as of 17:37, 27 October 2010

UT-Tokyo

Sudoku

Introduction System Modeling Lab note [ June / July / August / September - October ] Experiment Result Reference


Retrieved from "http://2010.igem.org/Team:UT-Tokyo/Sudoku_lab_note"