Team:Tokyo Tech/Project/Artificial Cooperation System/lux act rep

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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br>
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<td>2 Apple reporter<br>
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:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]
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:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]
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<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br>
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:3-1 ''lux'' activation/repression promoter  -YOU ARE HERE!-
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:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]
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:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]
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:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br>
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:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New series of P''ompC'']]
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:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]
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:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]
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</table>
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[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep/Pluxrep/assay1|Plux rep assay1(R0061)]]<br>
 
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[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep/Pluxrep/assay2|Plux rep assay2(K395008)]]<br>
 
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[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep/Pluxact/assay1|Plux act assay1(R0062)]]<br>
 
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[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep/Pluxact/assay2|Plux rep assay2(R0062)]]<br>
 
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<!--ここから書き始めて下さい-->
 
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=lux activation/repression part=
 
-
 
 
-
==I, characterization of R0061 (promoter repressed by LuxR/3OC6HSL)==
 
-
===Introduction===
 
-
We characterized luxR repression promoter. In the Artificial Cooperation System, we inserted chloramphenicol resistance coding sequence into this promoter. Thus, this promoter plays an important role in Artificial Cooperation System. We wanted to characterize the strength of this promoter which has never been done before in BioBrick in order to design new promoter based on this data.
 
-
First, we confirmed R0061, which is a existing BioBrick promoter repressed by LuxR/3OC6HSL complex. To confirm this promoter, we constructed following two plasmids (fig〇〇and fig〇〇)
 
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[[IMAGE:Tokyotech K395101.png|400px]]
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<div id="tf_SubWrapper">
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<font size="5"><b>3-1 ''lux'' activation/repression promoter</b></font>
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==Abstract==
 +
In Artificial Cooperation System, two types of cells use quorum sensing to recognize population of the counterpart and to help the counterparts when they are dying. The quorum sensing in this system is regulated by AHL dependent transcriptional activation/repression. Therefore, we characterized activation/repression promoters. We examined the existing LuxR repression promoter which has never been characterized before in BioBrick registry. Even though the GFP expression was repressed in the presence of AHL, cell-growth rate decreased because of the overexpression of GFP occurred in the absence of AHL. For this reason, we designed and constructed a new repression promoter that regulates the transcription appropriately dependent on the signal input.
 +
<br>
 +
 
 +
 
 +
[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1  LuxR activation promoter assay  (worked by Kitano Shohei & Eriko Uchikoshi)]]
 +
 
 +
[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|left|thumb|fig.3-1-3  LuxR repression promoter assay  (worked by Shohei Kitano & Eriko Uchikoshi)]]
 +
 
 +
==Introduction==
 +
 
 +
In Synthetic Biology, transcription activation is used frequently. Transcription repression by using AHL is also important, however, the device which has delay for transcription/translation through inverter is used a lot in this case. We decided to experience LuxR repression promoter for the quick response of signal dependent repression.<br>
 +
 
 +
==Result==
 +
===R0, characterization of R0062 (promoter activated by LuxR/3OC6HSL)===
 +
First, we characterized R0062, the well-known LuxR activation promoter in order to establish our Tokyo_Tech team experimental system for Artificial Cooperation System.<br>
 +
The expression of GFP with 100nM 3OC6HSL around 30-folds increased comparing with the expression without 3OC6HSL.<br>
 +
 
 +
[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1  LuxR activation promoter assay  (worked by Kitano Shohei & Eriko Uchikoshi)]]
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
We confirmed fluorescence intensity of LuxR activation promoter is dependent on 3OC6HSL concentration. The AHL concentration which shows half of maximam activity is less than 5nM. <br>
 +
[[IMAGE:tokyotech_LuxR ractivatio promoter assay2.jpg|400px|left|thumb|fig.3-1-2  Fluorescence intensity dependent on the concentration of AHL  (worked by Shohei Kitano & Eriko Uchikoshi)]]
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
===R1, characterization of R0061 & K395008 (promoter repressed by LuxR/3OC6HSL)===
 +
 
 +
[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|left|thumb|fig.3-1-3  LuxR repression promoter assay  (worked by Shohei Kitano & Eriko Uchikoshi)]]
 +
 
 +
 
 +
 
 +
 
 +
 
-
We introduced these two plasmid into DH5&alpha;.
 
-
===Result===
 
-
[[IMAGE:tokyotech_LuxR repression promoter assay(R0061).jpg|400px]]
 
-
In the presence of 3OC6HSL(100nM), the fluorescence is lower than in the absence of 3OC6HSL. We used a fusion of placIQ (I14032) to gfp (K121013) as a positive control and used promoterless gfp (K121013) as a negative control. We measured fluorescence by flow cytometry 3 hour after addition of 100nM 3OC6HSL.
 
-
===Conclution===
 
-
This ''E. coli'' expresses LuxR constitutively and has GFP under Plux rep, thus it is supposed that GFP expression is repressed when AHL exists. Fig〇〇 shows that R0061 works as we expected.
 
-
==II, designing the new promoters repressed by LuxR/3OC6HSL, K395008 and K395009==
 
-
Though R0061 is repressed by LuxR/3OC6HSL, the leaky expression is so high. That’s because -35 and -10 sequence of R0061 is the same as the -35 and -10 sequence of J23119 whose strength is the highest in BioBrick constitutive promoters.
 
-
[[IMAGE:Tokyotech J23119&R0061.png|400px]]
 
-
Then we designed new BioBrick parts, K395008 and K395009, whose -35 and -10 sequence is different from R0061.
 
-
The -35 and -10 sequence of K395008 is the same as that of J23108. The -35 and -10 sequence is the same as that of J23115. J23108 and J23115 are BioBrick constitutive promoters.
 
-
[[IMAGE:Tokyotech_R0061&K395008&K395009.png|400px]]
 
-
Why we chose J23108 and J23115 is that these expression levels are middle and low in BioBrick constitutive promoters. 2nd reason is that 3’ end nucleotide of -35 sequence of J23108 and J23115 is ‘A’ and that 5’ end nucleotide of -10 sequence 0f J23108 and J23115 is ‘T’. -35 and -10 overlap lux box.
 
-
[[IMAGE:Tokyotech_J23108&J23115.png|400px]]
 
-
==III, characterization of K395008 (LuxR repression promoter)==
 
-
===Introduction===
 
-
Even subtle changes in promoter may have distinct effects on the expression of gene. As we mentioned before, we designed a new promoter which is repressed by AHL and LuxR complex by changing one base of the existing BioBrick parts (BBa_R0061). We wanted to characterize this luxR repression promoter. Also, we wanted to confirm that this promoter is also repressed by AHL and LuxR but has different strength from the existing BioBrick part.
 
-
===Result===
 
-
After construction of K395195, we introduced K395195 and S03119 into DH5&alpha;. And we measured the fluorescence by flow cytometry. Fig 〇〇shows this result.
 
-
[[IMAGE:tokyotech_LuxR repression promoter assay(R0061weak).jpg|400px]]
 
-
Addition of AHL caused the decrease in fluorescence intensity. The expression of GFP with AHL dropped to 1/3 comparing with the expression without AHL.
 
-
===Conclusion===
+
===R1-1, R0061 (promoter repressed by LuxR/3OC6HSL)===
-
We confirmed that AHL repressed luxR repression promoter, K395008 as expected.
+
Next, we characterized the existing part R0061, LuxR repression promoter. We examined whether the amount of transcription is appropriate when signal is off and how much this promoter represses. <br>
 +
The expression of GFP with 100nM 3OC6HSL dropped to 1/3 comparing with the expression without 3OC6HSL.  
 +
===R1-2, K395008 (promoter repressed by LuxR/3OC6HSL):R0061 weak===
 +
We confirmed R0061 and found increase of cells was inhibited due to a high level of expression although it is repressed by AHL. Therefore, we designed a new appropriate promoter by changing one base of R0061.<br>
 +
The expression of GFP with 100nM 3OC6HSL dropped to 1/3 comparing with the expression without 3OC6HSL. We found the level of expression is appropriate and this promoter work as expected.
 +
==Conclusion==
 +
We designed the new promoter which is repressed LuxR/3OC6HSL complex by changing one base of existing promoter. We confirmed this promoter works as we expected.<br>
 +
It is not so difficult to make the promoter which strength is between these two by designing.
-
==IV, characterization of R0062 (promoter activated by LuxR/3OC6HSL)==
+
==Material & Methods==
 +
===M0, characterization of R0062 (promoter activated by LuxR/3OC6HSL)===
 +
===fluorescence intensity in the presence/absence of AHL ===
 +
We constructed K395100 combining R0062 and K121013. K121013 is a promoter-less ''gfp'' reporter (rbs-''gfp''-ter-ter) on pSB6A1. S03119 is a LuxR generator which is regulated by PTetR, which is repressed by TetR. In this experiment, we don’t use TetR, so S03119 functions as a LuxR constitutive generator. The backbone of S03119 is pSB1A2, which is a high copy plasmid, so we changed the backbone from pSB1A2 to pSB3K3.
 +
We used a fusion of PlacI<sup>q</sup> (I14032) to ''gfp'' (K121013) as a positive control and used promoterless ''gfp'' (K121013) as a negative control.
-
===Introduction===
+
[[IMAGE:Tokyotech_R0062assay_construction.png‎|400px]]
-
We characterized luxR activation promoter. We inserted chloramphenicol resistance coding sequence into the downstream of this promoter. This promoter plays an important role in Artificial Cooperation System. We wanted to confirm strength of this promoter which has already been done before in BioBrick in order to design new promoter based on this data.
+
-
First, we assayed R0062 which is an existing BioBrick promoter activated by LuxR/3OC6HSL complex. To confirm this promoter, we constructed following two plasmids (fig〇〇and fig〇〇)
+
-
[[IMAGE:Tokyotech K395100.png|400px]]
+
*samples
 +
#[Plux act - ''gfp''](BBa_K395100) on pSB6A1 + [PtetR – LuxR] on pSB3K3
 +
#positive control: [PlacI<sup>q</sup>(constitutive promoter) - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3 
 +
#negative control:. [promoterless - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3 
 +
*Strain
 +
DH5&alpha;
 +
*protocol
 +
#Prepare overnight culture.
 +
#Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan). (→fresh culture)
 +
#Incubate the fresh culture until the observed O.D. reaches around 0.60.
 +
#Each sample was divided into 2. Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other. The final concentration of 3OC6HSL is 100nM.
 +
#Induction for 3 hours at 37°C.
 +
#Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.
-
We introduced two plasmid into DH5&alpha;.  
+
====fluorescence intensity dependent on [AHL]====
-
Next, we measured the 3OC6HSL concentration dependence of Plux activity. We measured the fluorescence intensity under different concentration of AHL (0nM, 1nM, 3nM, 5nM, 10nM, 30nM, 50nM, 100nM. We measured the fluorescence by flow cytometry 3 hours after 3OC6HSL induction.
+
*samples
 +
#[Plux act - ''gfp''](BBa_K395100) on pSB6A1 + [ptet – LuxR] on pSB3K3
 +
*Strain
 +
DH5&alpha;
 +
*protocol
 +
#Prepare overnight culture.
 +
#Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan). (→fresh culture) Prepare the same 7 tubes for each sample.
 +
#Incubate the fresh culture until the observed O.D. reaches around 0.60.
 +
#Each sample was divided into 2. Prepare and add 3OC6HSL mixture. The final concentration of 3OC6HSL is 1, 3, 5, 10, 30, 50, 100nM.
 +
#Induction for 3 hours at 37°C.
 +
#Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.
-
===Result===
+
===M1, characterization of R0061 & K395008 (promoter repressed by LuxR/3OC6HSL)===
-
In the absence of AHL, the fluorescence is low. In the contrast, the fluorescence is so high in the presence of 3OC6HSL(100nM), that’s 〇〇fold higher than in the absence of 3OC6HSL.
+
====M1-1, characterization of R0061 (promoter repressed by LuxR/3OC6HSL)====
-
[[IMAGE:tokyotech_LuxR activation promoter assay(R0062).jpg|400px]]
+
We constructed K395101 combining R0061 and K121013, which is a promoter-less ''gfp'' reporter (rbs-''gfp''-ter-ter) on pSB6A1. S03119 is a LuxR generator which is repressed by TetR. In this experiment, we don’t use TetR, therefore, S03119 functions a LuxR constitutive generator. The backbone of S03119 is pSB1A2, which is a high copy plasmid, so we changed the backbone from pSB1A2 to pSB3K3.
 +
We used a fusion of PlacI<sup>q</sup> (I14032) to ''gfp'' (K121013) as a positive control and used promoterless ''gfp'' (K121013) as a negative control.
-
The previous experiment shows that Plux is worked. This means that Plux is activated by LuxR and 3OC6HSL.  
+
[[IMAGE:Tokyotech_R0061assay_construction.png‎|400px]]
-
[[IMAGE:tokyotech_LuxR ractivatio promoter assay2.jpg|400px]]
+
*'''samples'''
 +
#[Plux rep - ''gfp''](BBa_K395101) on pSB6A1 + [PtetR – LuxR] on pSB3K3
 +
#positive control: [PlacI<sup>q</sup>(constitutive promoter) - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3
 +
#negative control: [promoterless - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3
 +
*'''Strain'''
 +
DH5&alpha;
 +
*'''protocol'''
 +
#Prepare overnight culture.
 +
#Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan).(→fresh culture)
 +
#Incubate the fresh culture until the observed O.D. reaches around 0.80.
 +
#Each sample was divided into 2. Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other. The final concentration of AHL is 100nM.
 +
#Induction for 2 hours at 37°C.
 +
#Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.
-
Fig 〇〇is the result of measurement.
+
====M1-2, characterization of K395008 (promoter repressed by LuxR/3OC6HSL):R0061 weak====
-
===Conclusion===
+
We constructed K395105 combining K395008 and K121013. K121013 is a promoter-less ''gfp'' reporter (rbs-''gfp''-ter-ter) and this backbone is pSB6A1. Promoter of S03119 is PtetR, which is repressed by tetR. In this experiment, we don’t use TetR, so, S03119 functions a LuxR constitutive generator. The backbone of S03119 is pSB1A2, which is a high copy plasmid, so we changed the backbone from pSB1A2 to pSB3K3.
-
This ''E. coli'' expresses LuxR constitutively and has GFP under Plux act, thus it is supposed that GFP expression is activated when 3OC6HSL exists.
+
We used a fusion of PlacI<sup>q</sup> (I14032) to ''gfp'' (K121013) as a positive control and used promoterless ''gfp'' (K121013) as a negative control.
-
We confirmed BioBricks, K395100 and S03119 (pSB3K3) worked correctly and the activity of R0062 is dependent on 3OC6HSL and the AHL threshold concentration is about 〇〇nM.
+
 +
[[IMAGE:Tokyotech_K395008assay_construction.png‎|400px]]
 +
*samples
 +
#[R0061weak - ''gfp''](BBa_K395105) on pSB6A1 + [PtetR – LuxR] on pSB3K3
 +
#positive control: [PlacI<sup>q</sup>(constitutive promoter) - ''gfp''] on pSB6A1+ [PtetR – LuxR]) on pSB3K3
 +
#negative control: [promoterless - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3
 +
*Strain
 +
DH5&alpha;
 +
*protocol
 +
#Prepare overnight culture.
 +
#Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan). (→fresh culture)
 +
#Incubate the fresh culture until the observed O.D. reaches around 0.60.
 +
#Each sample was divided into 2. Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other. The final concentration of 3OC6HSL is 100nM.
 +
#Induction for 3 hours at 37°C.
 +
#Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.
 +
==Reference==
 +
#KRISTI A. EGLAND & E. P. GREENBERG, Conversion of the Vibrio fischeri Transcriptional Activator LuxR, to a Repressor. JOURNAL OF BACTERIOLOGY, Feb. 2000, p. 805–811

Latest revision as of 03:58, 28 October 2010

iGEM Tokyo Tech 2010 "E.coli with Humanity"

3-1 lux activation/repression promoter

Contents

Abstract

In Artificial Cooperation System, two types of cells use quorum sensing to recognize population of the counterpart and to help the counterparts when they are dying. The quorum sensing in this system is regulated by AHL dependent transcriptional activation/repression. Therefore, we characterized activation/repression promoters. We examined the existing LuxR repression promoter which has never been characterized before in BioBrick registry. Even though the GFP expression was repressed in the presence of AHL, cell-growth rate decreased because of the overexpression of GFP occurred in the absence of AHL. For this reason, we designed and constructed a new repression promoter that regulates the transcription appropriately dependent on the signal input.


fig.3-1-1 LuxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)
fig.3-1-3 LuxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)

Introduction

In Synthetic Biology, transcription activation is used frequently. Transcription repression by using AHL is also important, however, the device which has delay for transcription/translation through inverter is used a lot in this case. We decided to experience LuxR repression promoter for the quick response of signal dependent repression.

Result

R0, characterization of R0062 (promoter activated by LuxR/3OC6HSL)

First, we characterized R0062, the well-known LuxR activation promoter in order to establish our Tokyo_Tech team experimental system for Artificial Cooperation System.
The expression of GFP with 100nM 3OC6HSL around 30-folds increased comparing with the expression without 3OC6HSL.

fig.3-1-1 LuxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)












We confirmed fluorescence intensity of LuxR activation promoter is dependent on 3OC6HSL concentration. The AHL concentration which shows half of maximam activity is less than 5nM.

fig.3-1-2 Fluorescence intensity dependent on the concentration of AHL (worked by Shohei Kitano & Eriko Uchikoshi)













R1, characterization of R0061 & K395008 (promoter repressed by LuxR/3OC6HSL)

fig.3-1-3 LuxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)













R1-1, R0061 (promoter repressed by LuxR/3OC6HSL)

Next, we characterized the existing part R0061, LuxR repression promoter. We examined whether the amount of transcription is appropriate when signal is off and how much this promoter represses.
The expression of GFP with 100nM 3OC6HSL dropped to 1/3 comparing with the expression without 3OC6HSL.

R1-2, K395008 (promoter repressed by LuxR/3OC6HSL):R0061 weak

We confirmed R0061 and found increase of cells was inhibited due to a high level of expression although it is repressed by AHL. Therefore, we designed a new appropriate promoter by changing one base of R0061.
The expression of GFP with 100nM 3OC6HSL dropped to 1/3 comparing with the expression without 3OC6HSL. We found the level of expression is appropriate and this promoter work as expected.

Conclusion

We designed the new promoter which is repressed LuxR/3OC6HSL complex by changing one base of existing promoter. We confirmed this promoter works as we expected.
It is not so difficult to make the promoter which strength is between these two by designing.

Material & Methods

M0, characterization of R0062 (promoter activated by LuxR/3OC6HSL)

fluorescence intensity in the presence/absence of AHL

We constructed K395100 combining R0062 and K121013. K121013 is a promoter-less gfp reporter (rbs-gfp-ter-ter) on pSB6A1. S03119 is a LuxR generator which is regulated by PTetR, which is repressed by TetR. In this experiment, we don’t use TetR, so S03119 functions as a LuxR constitutive generator. The backbone of S03119 is pSB1A2, which is a high copy plasmid, so we changed the backbone from pSB1A2 to pSB3K3. We used a fusion of PlacIq (I14032) to gfp (K121013) as a positive control and used promoterless gfp (K121013) as a negative control.

Tokyotech R0062assay construction.png

  • samples
  1. [Plux act - gfp](BBa_K395100) on pSB6A1 + [PtetR – LuxR] on pSB3K3
  2. positive control: [PlacIq(constitutive promoter) - gfp] on pSB6A1+ [PtetR – LuxR] on pSB3K3
  3. negative control:. [promoterless - gfp] on pSB6A1+ [PtetR – LuxR] on pSB3K3
  • Strain

DH5α

  • protocol
  1. Prepare overnight culture.
  2. Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan). (→fresh culture)
  3. Incubate the fresh culture until the observed O.D. reaches around 0.60.
  4. Each sample was divided into 2. Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other. The final concentration of 3OC6HSL is 100nM.
  5. Induction for 3 hours at 37°C.
  6. Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.

fluorescence intensity dependent on [AHL]

  • samples
  1. [Plux act - gfp](BBa_K395100) on pSB6A1 + [ptet – LuxR] on pSB3K3
  • Strain

DH5α

  • protocol
  1. Prepare overnight culture.
  2. Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan). (→fresh culture) Prepare the same 7 tubes for each sample.
  3. Incubate the fresh culture until the observed O.D. reaches around 0.60.
  4. Each sample was divided into 2. Prepare and add 3OC6HSL mixture. The final concentration of 3OC6HSL is 1, 3, 5, 10, 30, 50, 100nM.
  5. Induction for 3 hours at 37°C.
  6. Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.

M1, characterization of R0061 & K395008 (promoter repressed by LuxR/3OC6HSL)

M1-1, characterization of R0061 (promoter repressed by LuxR/3OC6HSL)

We constructed K395101 combining R0061 and K121013, which is a promoter-less gfp reporter (rbs-gfp-ter-ter) on pSB6A1. S03119 is a LuxR generator which is repressed by TetR. In this experiment, we don’t use TetR, therefore, S03119 functions a LuxR constitutive generator. The backbone of S03119 is pSB1A2, which is a high copy plasmid, so we changed the backbone from pSB1A2 to pSB3K3. We used a fusion of PlacIq (I14032) to gfp (K121013) as a positive control and used promoterless gfp (K121013) as a negative control.

Tokyotech R0061assay construction.png

  • samples
  1. [Plux rep - gfp](BBa_K395101) on pSB6A1 + [PtetR – LuxR] on pSB3K3
  2. positive control: [PlacIq(constitutive promoter) - gfp] on pSB6A1+ [PtetR – LuxR] on pSB3K3
  3. negative control: [promoterless - gfp] on pSB6A1+ [PtetR – LuxR] on pSB3K3
  • Strain

DH5α

  • protocol
  1. Prepare overnight culture.
  2. Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan).(→fresh culture)
  3. Incubate the fresh culture until the observed O.D. reaches around 0.80.
  4. Each sample was divided into 2. Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other. The final concentration of AHL is 100nM.
  5. Induction for 2 hours at 37°C.
  6. Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.

M1-2, characterization of K395008 (promoter repressed by LuxR/3OC6HSL):R0061 weak

We constructed K395105 combining K395008 and K121013. K121013 is a promoter-less gfp reporter (rbs-gfp-ter-ter) and this backbone is pSB6A1. Promoter of S03119 is PtetR, which is repressed by tetR. In this experiment, we don’t use TetR, so, S03119 functions a LuxR constitutive generator. The backbone of S03119 is pSB1A2, which is a high copy plasmid, so we changed the backbone from pSB1A2 to pSB3K3. We used a fusion of PlacIq (I14032) to gfp (K121013) as a positive control and used promoterless gfp (K121013) as a negative control.

Tokyotech K395008assay construction.png

  • samples
  1. [R0061weak - gfp](BBa_K395105) on pSB6A1 + [PtetR – LuxR] on pSB3K3
  2. positive control: [PlacIq(constitutive promoter) - gfp] on pSB6A1+ [PtetR – LuxR]) on pSB3K3
  3. negative control: [promoterless - gfp] on pSB6A1+ [PtetR – LuxR] on pSB3K3
  • Strain

DH5α

  • protocol
  1. Prepare overnight culture.
  2. Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan). (→fresh culture)
  3. Incubate the fresh culture until the observed O.D. reaches around 0.60.
  4. Each sample was divided into 2. Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other. The final concentration of 3OC6HSL is 100nM.
  5. Induction for 3 hours at 37°C.
  6. Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.

Reference

  1. KRISTI A. EGLAND & E. P. GREENBERG, Conversion of the Vibrio fischeri Transcriptional Activator LuxR, to a Repressor. JOURNAL OF BACTERIOLOGY, Feb. 2000, p. 805–811






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