Team:Chiba/Experiments:copy number

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__NOTOC__
__NOTOC__
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==Copy number of Receiver Plasmid==
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==Balancing Player==
===Design===
===Design===
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[[Image:Receiver copy number chiba.jpg|left]]
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[[Image:Receiver copy number chiba.jpg|left|frame|'''Fig. 1　Player Balancing''']]
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レシーバーのコピーナンバーを変えることで、応答までの時間を変える<br>
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コピーナンバーを変えれば、レシーバーによるLuxRの合成量は変化する<br>
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-
AHLを受け取るLuxRが変わるので応答閾値までの時間が変わると考えた<br>
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-
<br clear=all>
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===Experiment===
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oriがcolE1のハイコピーベクターにのったBBa_T9002と、oriがP15Aのローコピーベクターに乗ったT9002バリエーションを使用した。
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これらを比較することでコピーナンバーを変えることでの影響を調べた。
 
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以下の遺伝子回路を使って、実験を行った。
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In this section, we intended to create time-delay by altering the
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===='''Sender'''====
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amount of luxR protein per cell.(*1) Since the amount of LuxR that
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*AHL autoinucer[http://partsregistry.org/Part:BBa_S03623 (BBa_S03623) ]
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receives AHL differs, we hypothesized that the time required to reach
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[[Image:LuxI-sender Chiba.gif|left|]]<br clear=all>
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the threshold AHL concentration will differ.
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===='''Receiver'''====
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To do this, we used different copy number plasmids of the Receivers. (*1)
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*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002 (Express GFP in response to AHL)]
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[[Image:High-Copy-Receiver Chiba.gif]]<br clear=all>
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By altering the copy number of plasmids in the receiver cells, the
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*Low Copy Receiver
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time required for the response is altered.<BR>
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[[Image:Low-Copy-Receiver Chiba.gif]]
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If the copy number is altered, the amount of LuxR produced by the
 +
receivers will also change.<BR>
 +
The amount of LuxR that receives AHL will change, so the time required
 +
for AHL to reach a threshold level will be altered.
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これらのほかにBBa_T9002にはChloramphenicol耐性マーカーを持つ空ベクター、Low copyバリエーションにはAmpicillin耐性マーカーの空ベクターをダブルトランスフォーメーションさせた。
 
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===Result & Discussion===
 
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[[Team:Chiba/AHL Receiver Phase#Plasmid Copy number|more about Plasmid Copy number]]<br>
 
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[[Image:Copynomber-change-result Chiba.gif|thumb|left|'''Fig.''' Time Delay Test]]
 
<br clear=all>
<br clear=all>
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*クオラムセンシングに関わる遺伝子のベクタープラスミドのコピーナンバーを少なくすることで、遺伝子発現が遅くなる
 
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*それと同時に、遺伝子発現の最大値自体も少なくなってしまう
 
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[[Team:Chiba/AHL Receiver Phase#Plasmid_Copy_number|more about experimental result]]
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===Experiment===
 +
We used a pMB1 ori high copy vector containing BBa_T9002 and a low
 +
copy vector containing BBa_T9002 with a P15A as ori.
 +
We determined the effects of altering the plasmid copy number by
 +
comparing the above situations.
 +
 +
Experiments used the following genetic circuits:
<br clear=all>
<br clear=all>
 +
{| class="tbl" |
 +
|-
 +
|
 +
*'''Sender'''
 +
|
 +
*'''Receivers'''
 +
|-
 +
| rowspan="2" | **[http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL autoinucer)]
 +
[[Image:LuxI-sender Chiba.gif]]
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|
 +
**[http://partsregistry.org/Part:BBa_T9002 BBa_T9002 (Express GFP in response to AHL)] |
 +
[[Image:High-Copy-Receiver Chiba.gif]]
 +
|-
 +
|
 +
*Low Copy Receiver
 +
[[Image:Low-Copy-Receiver Chiba.gif]]
 +
|}
 +
<br clear=all>
 +
In addition, a Chloramphenicol resistance marker-containing empty
 +
plamid was transformed to the strain containing BBa_T9002, and a
 +
Ampicillin resistance marker-containing empty vector was transformed
 +
into the low copy variant, both resulting in a double transformation.
 +
<br clear=all>
===Method===
===Method===
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#Transformed sender(Ptet-LuxI), high copy receiver(Ptet-LuxR-Plux-GFP-colE1) and Medium copy receiver(Ptet-LuxR-Plux-GFP-p15A) respectively into E coli strains(BD⊿FliC).
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#Transformed sender (Ptet-LuxI), high copy receiver (Ptet-LuxR-Plux-GFP-pMB1) and Medium copy receiver (Ptet-LuxR-Plux-GFP-p15A) respectively into ''E coli'' strains (JW1908).
#Inoculated them independently in liquid media. Incubated at 37&deg;C 12h.
#Inoculated them independently in liquid media. Incubated at 37&deg;C 12h.
#Inoculated again in Fresh liquid media upto about OD600=2 at 37&deg;C
#Inoculated again in Fresh liquid media upto about OD600=2 at 37&deg;C
#Washed sender and receivers.
#Washed sender and receivers.
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#Mixed them. (Sender:Receiver=1000μL:1000μL)
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#Mixed them. (Sender:Receiver=1000&mu;L:1000&mu;L)
#Incubated at 30&deg;C.
#Incubated at 30&deg;C.
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#Measured intensity of green fluorescence at regular time intervals.(Fluoroskan AscentR FL＆Fluoroskan AscentR　Thermo ELECTRON CORPORATION)
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#Measured intensity of green fluorescence at regular time intervals. (Fluoroskan AscentR FL&Fluoroskan AscentR　Thermo Electron Corporation)
 +
 
 +
===Result & Discussion===
 +
[[Image:Copy-nomber-change-result-detail version Chiba.gif|frame|left|'''Fig. 2　Time Delay Test: Highcopy Receiver & Reporter vs.Lowcopy Receiver & Reporter''']]
 +
<br clear=all>
 +
 
 +
 
 +
Compared to GFP expression from (Ptet-LuxR-plux-GFP-pMB1), expression
 +
from (Ptet-LuxR-pLux-GFP-p15A) was greatly reduced (Fig. 2).
 +
 
 +
Possible explanations for this are:
 +
<BR>
 +
#GFP expression was reduced since it was placed on a low copy number plasmid.
 +
#The amount of LuxR synthesized was too small.
 +
However, (Ptet-luxR-p15A + plux-GFP-pMB1) and
 +
(Ptet-LuxR-pLux-GFP-pMB1) resulted in exactly the same trasnfer Curve
 +
(Fig. 3).
 +
 
 +
Thus, we can deduce that the expression levels of LuxR from the p15A
 +
vector is sufficient to activate pLux from the high copy number
 +
vector. Explanation 1 is therefore more probable.
 +
 
 +
[[Image:LuxR-high-Low-result Chiba.gif|frame|left|'''Fig. 3　Time Delay Test: Lowcopy Receiver & Highcopy Reporter vs.Highcopy Receiver-Reporter''']]
 +
 
 +
 
 +
 
 +
 
 +
<br clear=all>
 +
 
 +
=== Demo ~Receivers~ ===
 +
Breif explanation on Methods:<br>
 +
1. LB pre-cultured Sender (BBa_S03623 (JW1908)) was mixed with LB-agar to produce sender containing bacterial plate.<br>
 +
2. Receiver colony was transfered to a nitrocellulose filter and placed on a Sender (BBa_S03623 (JW1908)) containing bacterial plate.<br>
 +
3. AHL produced by sender is diffused in the LB-agar plate and the receivers on the nitrocellulose filter synthesize GFP when they are stimulated by a certain amount of AHL.<br>
 +
4. Plates cultured at 37°C were exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence. <br>
 +
 
 +
The receivers are:<br>
 +
*AiiA synthesizing receiver.<br>
 +
*receiver which has different copy number.<br>
 +
*Cross-talk receiver.<br>
 +
 
 +
 
 +
====Results====
 +
 
 +
--->more about [[Team:Chiba/Demo_experiments#Demo Experiment ~Receivers~|Receivers Demo experiments detail]]
 +
 
 +
 
 +
<br clear=all>
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'''>[[Team:Chiba/Project#Receiver|Back to the project page]]'''
 +
<br clear=all>
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{| style="color:white;background-color:Maroon" cellpadding="3" cellspacing="3" border="1" bordercolor="white" width="100%" align="center"
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!align="center"|[[Team:Chiba|Home]]
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!align="center"|[[Team:Chiba/Team|The Team]]
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!align="center"|[[Team:Chiba/Project|The Project]]
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!align="center"|[[Team:Chiba/Parts|Parts Submitted to the Registry]]
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!align="center"|[[Team:Chiba/Notebook|Notebook]]
 +
|}

Latest revision as of 09:37, 30 October 2008

Chiba-U.gif
Home The Team The Project Parts Submitted to the Registry Reference Notebook Acknowledgements


Balancing Player

Design

Fig. 1　Player Balancing


In this section, we intended to create time-delay by altering the amount of luxR protein per cell.(*1) Since the amount of LuxR that receives AHL differs, we hypothesized that the time required to reach the threshold AHL concentration will differ.

To do this, we used different copy number plasmids of the Receivers. (*1)

By altering the copy number of plasmids in the receiver cells, the time required for the response is altered.
If the copy number is altered, the amount of LuxR produced by the receivers will also change.
The amount of LuxR that receives AHL will change, so the time required for AHL to reach a threshold level will be altered.



Experiment

We used a pMB1 ori high copy vector containing BBa_T9002 and a low copy vector containing BBa_T9002 with a P15A as ori.

We determined the effects of altering the plasmid copy number by comparing the above situations.

Experiments used the following genetic circuits:

  • Sender
  • Receivers
**[http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL autoinucer)]

LuxI-sender Chiba.gif

    • [http://partsregistry.org/Part:BBa_T9002 BBa_T9002 (Express GFP in response to AHL)] |

High-Copy-Receiver Chiba.gif

  • Low Copy Receiver

Low-Copy-Receiver Chiba.gif



In addition, a Chloramphenicol resistance marker-containing empty plamid was transformed to the strain containing BBa_T9002, and a Ampicillin resistance marker-containing empty vector was transformed into the low copy variant, both resulting in a double transformation.


Method

  1. Transformed sender (Ptet-LuxI), high copy receiver (Ptet-LuxR-Plux-GFP-pMB1) and Medium copy receiver (Ptet-LuxR-Plux-GFP-p15A) respectively into E coli strains (JW1908).
  2. Inoculated them independently in liquid media. Incubated at 37°C 12h.
  3. Inoculated again in Fresh liquid media upto about OD600=2 at 37°C
  4. Washed sender and receivers.
  5. Mixed them. (Sender:Receiver=1000μL:1000μL)
  6. Incubated at 30°C.
  7. Measured intensity of green fluorescence at regular time intervals. (Fluoroskan AscentR FL&Fluoroskan AscentR　Thermo Electron Corporation)

Result & Discussion

Fig. 2　Time Delay Test: Highcopy Receiver & Reporter vs.Lowcopy Receiver & Reporter



Compared to GFP expression from (Ptet-LuxR-plux-GFP-pMB1), expression from (Ptet-LuxR-pLux-GFP-p15A) was greatly reduced (Fig. 2).

Possible explanations for this are:

  1. GFP expression was reduced since it was placed on a low copy number plasmid.
  2. The amount of LuxR synthesized was too small.

However, (Ptet-luxR-p15A + plux-GFP-pMB1) and (Ptet-LuxR-pLux-GFP-pMB1) resulted in exactly the same trasnfer Curve (Fig. 3).

Thus, we can deduce that the expression levels of LuxR from the p15A vector is sufficient to activate pLux from the high copy number vector. Explanation 1 is therefore more probable.

Fig. 3　Time Delay Test: Lowcopy Receiver & Highcopy Reporter vs.Highcopy Receiver-Reporter




Demo ~Receivers~

Breif explanation on Methods:
1. LB pre-cultured Sender (BBa_S03623 (JW1908)) was mixed with LB-agar to produce sender containing bacterial plate.
2. Receiver colony was transfered to a nitrocellulose filter and placed on a Sender (BBa_S03623 (JW1908)) containing bacterial plate.
3. AHL produced by sender is diffused in the LB-agar plate and the receivers on the nitrocellulose filter synthesize GFP when they are stimulated by a certain amount of AHL.
4. Plates cultured at 37°C were exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.

The receivers are:

  • AiiA synthesizing receiver.
  • receiver which has different copy number.
  • Cross-talk receiver.


Results

--->more about Receivers Demo experiments detail



>Back to the project page

Home The Team The Project Parts Submitted to the Registry Notebook
Retrieved from "http://2008.igem.org/Team:Chiba/Experiments:copy_number"