Team:Paris/August 17

From 2008.igem.org

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(→PCR Screening)
(→Construction of OmpR*+RBS and EnvZ*+RBS: transformation)
 
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|3
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|Problem
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|L151
|L151
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|21
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|colspan="6" |Controls
|colspan="6" |Controls
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|150
|NO
|NO
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|OK
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|style="background: #cbff7B"|OK
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|C2
|C2
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|2
|No
|No
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|OK
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|style="background: #cbff7B"|OK
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|Positive Control
|Positive Control
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|416 (efficiency 4.10^7)
|416 (efficiency 4.10^7)
|No
|No
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|OK
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  Conclusion :  
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  Conclusion : <br>L150 doesn't success, measured size match with the promoter size only, <br> we will check the size of L101 by screening and if it doesn't match with the good size (1827pb) <br> we will try again the ligation : rbs-TetR (S03879) with GFP tripart (E0840).<br>L151 is ok we have to put a strong promoter before the part rbs-lasR-double terminator :GREAT !
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L150 doesn't success, measured size match with the promoter size only, we will check the size of L101 by screening and if it doesn't match with the good size (1827pb) we will try again the ligation : rbs-TetR (S03879) with GFP tripart (E0840).<br>L151 is ok we have to put a strong promoter before the part rbs-lasR-double terminator :GREAT !
+
 
 +
===Minipreps & Stocks===
 +
* Cultured in 7,5ml of LB with a thoothpick of a colony.
 +
* Culture O/N at 37°C of :
-
===Minipreps===
 
{|border="1" style="text-align: center"
{|border="1" style="text-align: center"
|'''Miniprep Name'''
|'''Miniprep Name'''
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=Creation of a registry of pFliL, pFlhDC, and ''FlhDC''=
=='''Analysis of the other transformation we did [[Team:Paris/August 16 |yesterday]]'''==
=='''Analysis of the other transformation we did [[Team:Paris/August 16 |yesterday]]'''==
===Evaluation of the number of colonies===
===Evaluation of the number of colonies===
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|>5000
|>5000
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  Remarks:
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  Remarks: <br>
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     L143 and L144 did not work once again. Maybe there is a problem with the digestion, because the primers used present only  
+
     L143 and L144 did not work once again. Maybe there is a problem with the digestion, <br> because the primers used present only two nucleotides after the restriction sites. what we should <br>do is cutting pfliL with xbaI and SpeI. As the vector will autoligate, we should use a phosphatase <br> to prevent this phenomenon. <br>
-
two nucleotides after the restriction sites. what we should do is cutting pfliL with xbaI and SpeI. As the vector will
+
     For L147 and its control (T4), we do not observe any red fluorescence. It means that the digestion work well, <br>because if it was not digested, the strong promoter J23100 should express mRFP! <br> '''We will repeat all the ligation anyway'''.
-
autoligate, we should use a phosphatase to prevent this phenomenon.
+
-
     For L147 and its control (T4), we do not observe any red fluorescence.
+
-
    It means that the digestion work well, because if it was not digested, the strong promoter J23100 should express mRFP!  
+
-
    We will do the screening anyway.
+
-
 
+
-
===PCR Screening===
+
-
===Minipreps===
+
-
=='''Construction of OmpR*+RBS and EnvZ*+RBS: transformation'''==
+
=Construction of OmpR*+RBS and EnvZ*+RBS: transformation=
===Evaluation of the number of colonies===
===Evaluation of the number of colonies===

Latest revision as of 18:24, 4 September 2008

← Yesterday

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Tomorrow →

Contents

Construction of pLas-TetR-GFP tripart & rbs-LasR-dble ter

Analysis of the transformation we did yesterday

Number of colonies and flurorescence

Ligation name Description Antibio Number Colonies observed Fluorescence Comments
Ligations
L150 D105(BV) - D146(BI)
pLas - rbs-TetR-GFP Tripart 		Amp 3 No Problem
L151 D147(FI) - D125(FV)
rbs-LasR - Double terminator 		Kan 21 No OK
Controls
C1 D105(BV) Amp 150 NO OK
C2 D125(FV) Kan 2 No OK
Positive Control pUC19 Amp 416 (efficiency 4.10^7) No OK

PCR Screening

Protocol

L150 and L151
Well Sample Expected size Measured size
1 1kb ladder
2 Negative control (pUC19) nothing nothing
3 Positive control (MP101.1) 768 pb 800pb
4 L150.1 1992pb 400pb
5 L150.2
6 L150.3
7 L151.1 1229pb 1200pb
8 L151.2
9 L151.3
10 L151.4
11 L151.5
12 L151.6
13 L151.7
14 L151.8
15 100pb ladder 
Conclusion : 
L150 doesn't success, measured size match with the promoter size only, 
 we will check the size of L101 by screening and if it doesn't match with the good size (1827pb) 
 we will try again the ligation : rbs-TetR (S03879) with GFP tripart (E0840).
L151 is ok we have to put a strong promoter before the part rbs-lasR-double terminator :GREAT !

Minipreps & Stocks

  • Cultured in 7,5ml of LB with a thoothpick of a colony.
  • Culture O/N at 37°C of :
Miniprep Name Ligation name Antibio Biobricks Description
MP161.1 L150.1 Amp Part icon regulatory.pngPart icon rbs.pngIcon coding.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png pLas - rbs-TetR-GFP tripart
MP161.2 L150.2
MP161.3 L150.3
MP162.1 L151.1 Kan Part icon rbs.pngIcon coding.pngPart icon terminator.pngPart icon terminator.png rbs-LasR - Double terminator
MP162.2 L151.2
MP162.3 L151.3

Creation of a registry of pFliL, pFlhDC, and FlhDC

Analysis of the other transformation we did yesterday

Evaluation of the number of colonies

Ligation name Number of colonies Autoligation control
L 143 (Kan) 0 0
L 144 (Kan) 0 0
L 145 (Amp) 452 430
L 146 (Amp) ~ 2000 430
L 147 (Amp) ~3500 >5000 
Remarks: 

    L143 and L144 did not work once again. Maybe there is a problem with the digestion,  
 because the primers  used present only two nucleotides after the restriction sites. what we should  
do is cutting pfliL with xbaI and SpeI. As the vector will autoligate, we should use a phosphatase  
 to prevent this phenomenon. 

    For L147 and its control (T4), we do not observe any red fluorescence. It means that the digestion work well, 
because if it was not digested, the strong promoter J23100 should  express mRFP!  
 We will repeat all the ligation anyway.

Construction of OmpR*+RBS and EnvZ*+RBS: transformation

Evaluation of the number of colonies

Ligation name Number of colonies Autoligation control
L 148 (Amp) ~10000 ~2000
L 149 (Amp) ~750 ~2000
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