Team:Chiba/lux promoter

From 2010.igem.org

(Difference between revisions)

Latest revision as of 13:29, 24 January 2011

Abstract

LuxR is an AHL-dependent activator.LuxR-AHL complex bind lux box,a 20-bp sequence and activate transcription. However the lux box is inserted between -35 and -10 and LuxR functions as LuxR-AHL inverter (Plux inv) .The Plux inv was resistered in Biobrick number R0061.We've constructed Plux inv-GFP combinig R0061and E0240 and characterized about it.

Experiments

1.Preparing Plux inv-GFP

Plasmid:R0061 and E0240
Strain:XL10-G
protocol
Preparing process of Plux inv-GFP is shown in Fig. 1.We transformed preparing Plux inv-GFP in XL10-G and confirmed the expression GFP Fluorescence.The sequence is also conformed.

Fig. 1 　Evaluation of LuxR Inverter

2.Characterization Plux inv-GFP

Plasmid:Plux inv-GFP and Plac-LuxR (LuxR constitutive generator)
strain:DH10B

protocol
We cotransformded Plux inv-GFP and LuxR Plac-LuxR in DH10B. 1,LBL incubation for12 h at 37゜C.

Results

We confirmed GFP Fluorescence both AHL+ and -sample.

Fig. 3

discussion

私たちの系では，LuxRの抑制を確認することができなかった。

Reference

Egland.K.A, and Greenberg.E.P, Conversion of the Vibrio Fischeri Transcriptional Activator,LuxR, to a Repressor, J. Bacteriol., 182, P.805-811 (2000)
Cox.R.S.3rd, Surette.M.G, Elowitz.M.B, Programming gene expression with combinatorial promoters, Mol Syst Biol, 3 ,145 (2007)

@@ Line 151: / Line 151: @@
 ==Abstract==
-BBa_R0061は-35および-10の間にlux box を含む配列である。継続的に下流遺伝子の転写を行う。しかしながら，LuxR-AHL複合体によって下流の遺伝子転写を抑制する。<br>
-私たちは，Plux invの下流にRBSおよびGFP遺伝子を挿入したプラスミド (Plux inv-GFP) を作製し，抑制機能を確認した。<br>
-Biobrick number R0061 contains lux box between -35 and -10  and fuctions LuxR-AHL inverter.
-We've prepare Plux inv-GFP and characterized about it.
+LuxR is an AHL-dependent activator.LuxR-AHL complex bind lux box,a 20-bp sequence and activate transcription.  However the lux box is inserted between -35 and -10 and LuxR functions as LuxR-AHL inverter (Plux inv) .The Plux inv was resistered in Biobrick number R0061.We've constructed Plux inv-GFP combinig R0061and E0240 and characterized about it.<br>
 ==Experiments==
 ===1.Preparing Plux inv-GFP===
-．Plux inv－GFPの作製
-クローニングのprocessをFig. 1に示す。作製したPlux inv-GFPを形質転換した株は緑色蛍光を示した。また，sequenceも正しいことを確認した。
+Plasmid:R0061 and E0240<br>
-Preparing Plux inv-GFP process is shown in '''Fig. 1'''.
+Strain:XL10-G<br>
+protocol<br>
+Preparing process of Plux inv-GFP  is shown in '''Fig. 1'''.We transformed preparing Plux inv-GFP in XL10-G and confirmed the expression GFP Fluorescence.The sequence is also conformed.
 [[Image:Plux inv-GFP.png|none|frame|right|'''Fig. 1''' 　Evaluation of LuxR Inverter]]
 ===2.Characterization Plux inv-GFP===
-Plux inv-GFP およびLuxR generatorである Plac-LuxRをDH10B株に共形質転換した。コロニーをつついてLB液体培地(AHL0および1000 nM)で12 h培養し,
-スピンダウンしてペレットを観察した。
+Plasmid:Plux inv-GFP and Plac-LuxR (LuxR constitutive generator)<br>
-[[Image:lux inverter function.png|frame|center|Fig. 2 assay protocol]]
+strain:DH10B<br>
+protocol<br>
+We cotransformded Plux inv-GFP and LuxR Plac-LuxR in DH10B.
+,LBL
+incubation for12 h at 37゜C.
 ==Results==
-[[Image:Plux inv results.png|none|right|Fig. 3 ]]
+We confirmed GFP Fluorescence both AHL+ and -sample.<br>
+<br>
+[[Image:Plux inv results.png|frame|none|Fig. 3 ]]
+==discussion==
+私たちの系では，LuxRの抑制を確認することができなかった。
 ==Reference==
 #Egland.K.A, and Greenberg.E.P, Conversion of the ''Vibrio Fischeri'' Transcriptional Activator,LuxR, to a Repressor, ''J. Bacteriol.'', '''182''', P.805-811 (2000)
 #Cox.R.S.3rd, Surette.M.G, Elowitz.M.B, Programming gene expression with combinatorial promoters, ''Mol Syst Biol'', '''3''' ,145 (2007)