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

From 2010.igem.org

(Difference between revisions)
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<div id="tf_SubWrapper">
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<font size="5"><b>3-1 ''lux'' activation/repression promoter</b></font>
+
=’lux’ activation/repression promoter=
-
==I, characterization of R0061 (promoter repressed by LuxR/3OC6HSL)==
+
=Abstract=
-
===Introduction===
+
In Artificial Cooperation System, two types of cells use cell-to-cell communication to recognize population of the counterpart and help the other when one is dying. The QS in this cell-cell communication is regulated by 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 and found that the growth of cells was inhibited when cells produce large amount of GFP even it is repressed by AHL. For this reason, we designed the promoter transcripts appropriately when signal is on/off.<br>
-
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.
+
=Introduction=
-
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)
+
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 of all, we characterized well known luxR activation promoter, R0062, in order to establish our Tokyo_Tech tram experimental system for Artificial Cooperation System.<br>
 +
The expression of GFP with 100nM 3OC6HSL around 30 holds increased comparing with the expression without 3OC6HSL.<br>
-
[[IMAGE:Tokyotech K395101.png|400px]]
+
[[IMAGE:Tokyotech_plux_act1_final.jpg|400px]]
-
We introduced these two plasmid into DH5&alpha;.
+
We confirmed fluorescence intensity of luxR activation promoter is dependent on 3OC6HSL concentration. The threshold of fluorescence intensity of R0062, luxR activation promoter regulated by 3OC6HSL is around 5nM.<br>
-
===Result===
+
[[IMAGE:tokyotech_LuxR ractivatio promoter assay2.jpg|400px]]
-
[[IMAGE:tokyotech_LuxR repression promoter assay(R0061).jpg|400px]]
+
==R1, characterization of 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. <br>
 +
The expression of GFP with 100nM 3OC6HSL dropped to 1/3 comparing with the expression without 3OC6HSL.  
-
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.
+
[[IMAGE:Tokyotech_plux_rep1_final.jpg|400px]]
-
===Conclution===
+
==R2, characterization of K395008 (promoter repressed by LuxR/3OC6HSL)==
-
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.  
+
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>
-
[https://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep/Pluxrep/assay1 (see more about Plux rep assay1...)]
+
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.
-
==II, designing the new promoters repressed by LuxR/3OC6HSL, K395008 and K395009==
+
[[IMAGE:Tokyotech_plux_rep2_final.jpg|400px]]
-
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]]
+
=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.
-
Then we designed new BioBrick parts, K395008 and K395009, whose -35 and -10 sequence is different from R0061.
+
=Material & Methods=
-
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.
+
==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.  
-
[[IMAGE:Tokyotech_R0061&K395008&K395009.png|400px]]
+
[[IMAGE:Tokyotech_R0062assay_construction.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.
+
*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.
-
[[IMAGE:Tokyotech_J23108&J23115.png|400px]]
+
===fluorescence intensity dependent on [AHL]===
 +
*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.
 +
==M1, 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 PlacI<sup>q</sup> (I14032) to gfp (K121013) as a positive control and used promoterless gfp (K121013) as a negative control.
-
==III, characterization of K395008 (LuxR repression promoter)==
+
[[IMAGE:Tokyotech_R0061assay_construction.png‎|400px]]
-
===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===
+
*'''samples'''
-
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.
+
#[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.
 +
==M2, characterization of K395008 (promoter repressed by LuxR/3OC6HSL)==
 +
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 PlacI<sup>q</sup> (I14032) to gfp (K121013) as a positive control and used promoterless gfp (K121013) as a negative control.
-
[[IMAGE:tokyotech_LuxR repression promoter assay(R0061weak).jpg|400px]]
+
[[IMAGE:Tokyotech_K395008assay_construction.png‎|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===
+
-
We confirmed that AHL repressed luxR repression promoter, K395008 as expected.
+
-
[https://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep/Pluxrep/assay2 (see more about Plux rep asssay2...)]
+
-
 
+
-
==IV, characterization of R0062 (promoter activated by LuxR/3OC6HSL)==
+
-
 
+
-
===Introduction===
+
-
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]]
+
-
 
+
-
We introduced two plasmid into DH5&alpha;.
+
-
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.
+
-
 
+
-
===Result===
+
-
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.
+
-
 
+
-
[[IMAGE:tokyotech_LuxR activation promoter assay(R0062).jpg|400px]]
+
-
 
+
-
The previous experiment shows that Plux is worked. This means that Plux is activated by LuxR and 3OC6HSL.
+
-
 
+
-
[[IMAGE:tokyotech_LuxR ractivatio promoter assay2.jpg|400px]]
+
-
Fig 〇〇is the result of measurement.
+
*samples
-
===Conclusion===
+
#[R0061weak - GFP](BBa_K395105) on pSB6A1 + [PtetR – LuxR] on 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.
+
#positive control: [PlacI<sup>q</sup>(constitutive promoter) - GFP] on pSB6A1+ [PtetR – LuxR]) on pSB3K3
-
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.
+
#negative control: [promoterless - GFP] on pSB6A1+ [PtetR – LuxR] on pSB3K3
-
[https://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep/Pluxact/assay1 (see more about Plux act assay1...)]
+
*Strain
-
[https://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep/Pluxact/assay2 (see more about Plux act assay2...)]
+
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.

Revision as of 14:22, 27 October 2010

iGEM Tokyo Tech 2010 "E.coli with Humanity"

Contents

’lux’ activation/repression promoter

Abstract

In Artificial Cooperation System, two types of cells use cell-to-cell communication to recognize population of the counterpart and help the other when one is dying. The QS in this cell-cell communication is regulated by 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 and found that the growth of cells was inhibited when cells produce large amount of GFP even it is repressed by AHL. For this reason, we designed the promoter transcripts appropriately when signal is on/off.

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

Tokyotech plux act1 final.jpg

We confirmed fluorescence intensity of luxR activation promoter is dependent on 3OC6HSL concentration. The threshold of fluorescence intensity of R0062, luxR activation promoter regulated by 3OC6HSL is around 5nM.

Tokyotech LuxR ractivatio promoter assay2.jpg

R1, characterization of 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.

Tokyotech plux rep1 final.jpg

R2, characterization of K395008 (promoter repressed by LuxR/3OC6HSL)

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.

Tokyotech plux rep2 final.jpg

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.

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

DH5&alpha

  • 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

DH5&alpha

  • 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 (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

DH5&alpha

  • 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.

M2, characterization of K395008 (promoter repressed by LuxR/3OC6HSL)

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

DH5&alpha

  • 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.








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