Team:ZJU-China/Project/Standardization/Results

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== Standardization: Results ==
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== Characterization: Results ==
__TOC__
__TOC__
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=== Standardization of Theophylline Riboswitch (BBa_K411003) ===
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=== Characterization of Theophylline Riboswitch (BBa_K411003) ===
We choose BBa_K411003, a typical riboswitch used by many iGEM teams, to be our first riboswitch to be standardized, which was firstly designed by iGEM10_NYMU-Taipei and has been tested by ZJU_China 2012.
We choose BBa_K411003, a typical riboswitch used by many iGEM teams, to be our first riboswitch to be standardized, which was firstly designed by iGEM10_NYMU-Taipei and has been tested by ZJU_China 2012.
-
Protocol:
+
<center>
-
# Cultivate the BL21 containing BBa_K411003 in LB at 37℃180rpm until OD600 reaches 0.5.
+
[[File:ZJU-Standardization 1.2.jpg|500px]]
-
# Induce the bacteria with 1mM IPTG (final concentration), and cultivate them at 37℃180rpm for 4hrs.
+
 
-
# Dilute the suspension culture with LB to 0.1OD600 . Theophylline is added at concentration ranging from 1.00E-05 mol/L to1.00E-01 mol/L.
+
Fig 1.1 shows how the whole GFP Change Rate(GCR) changes at different time and under different concentration. Initially, the culture induced by 0.5M Theophylline has the largest whole GCR. And almost every culture’s GCR experiences decrement along time.
-
# Measure the RFU ( ex488/em525) and OD600 synchronously.
+
</center>
-
# Data Analysis
+
 
<center>
<center>
-
[[File:ZJU-Standardization 1.1.jpg|500px]]
+
[[File:ZJU-Standardization 1.3.jpg|500px]]
-
Fig 1.1  the RFU/OD decreases along the time which we believe the rapid growth of OD has contributed most.
+
Fig 1.2 GFP Change Rate versus Concentration at 100 minutes after Theophylline was added.
</center>
</center>
 +
<center>
<center>
-
[[File:ZJU-Standardization 1.2.jpg|500px]]
+
[[File:Input_compatibility_of_411003.png|500px]]
-
Fig 1.2 shows how the whole GFP Change Rate(GCR) changes at different time and under different concentration. Initially, the culture induced by 0.5M Theophylline has the largest whole GCR. And almost every culture’s GCR experiences decrement along time.
+
Fig 1.3 Input Compatibility of K411003
</center>
</center>
 +
It can be easily observed that the riboswitch’s response to Theophylline is much stronger than to  the analogs of theophylline ,including diprophylline, cocaine, caffeine, hypoxanthine and xanthine.
<center>
<center>
-
[[File:ZJU-Standardization 1.3.jpg|500px]]
+
[[File:K411003_high.png|500px]]
-
Fig 1.3 GFP Change Rate versus Concentration at 100 minutes after Theophylline was added.
+
Fig 1.4 Performance Reliability of K411003 (Cultivated with Theophylline)
</center>
</center>
-
=== Standardization of Atrazine Riboswitch (BBa_1054008) ===
+
<center>
-
Since our project Atrazine Elf is based on the Atrazine-specific Riboswitch , our second choice to be standardized is Atrazine Riboswitch.
+
[[File:K411003_low2.png|500px]]
-
Protocol:
+
Fig 1.5 Performance Reliability of K411003 (Cultivated with NO Theophylline)
-
# Cultivate the BL21 containing BBa_ in LB at 37℃180rpm until OD600 reaches 0.5.
+
</center>
-
# Induce the bacteria with 1mM IPTG (final concentration), and cultivate them at 37℃180rpm for 4hrs.
+
 
-
# Dilute the suspension culture with LB to 0.1OD600 . Theophylline is added at concentration ranging from 1.00E-06 mol/L to1.00E-01 mol/L.
+
It obvious that the K411003 riboswitch cannot response correctly to theophylline after being cultivated and diluted for 4 days.
-
# Measure the RFU ( ex488/em525) and OD600 synchronously.
+
 
-
# Data Analysis
+
=== Characterization of Atrazine Riboswitch  (BBa_K1054008) ===
 +
Our second choice to be standardized is Atrazine Riboswitch.
 +
 
 +
<center>
 +
[[File:Static_of_Atrazine.png|500px]]
 +
 
 +
Fig 2.1 Static Performance of Atrazine Riboswitch
 +
</center>
<center>
<center>
[[File:ZJU-Standardization 2.1.jpg|500px]]
[[File:ZJU-Standardization 2.1.jpg|500px]]
-
Fig 2.1 Graphic Model about how RFU/OD changes at different time and different concentration of Atrzine.
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Fig 2.2 Graphic Model about how RFU/OD changes at different time and different concentration of Atrzine.
</center>
</center>
<center>
<center>
-
[[File:ZJU-Standardization 2.2.jpg|500px]]
+
[[File:Input_of_atrazine.jpg|500px]]
-
Fig 2.2 Graphic Model about how GFP Change Rate changes at different time and different concentration of Atrzine.
+
Fig 2.3 Input Compatibility of Atrazine.
</center>
</center>
-
=== Standardization of Theophylline Riboswitch 2(BBa_1054022 ) ===
+
=== Characterization of Theophylline Riboswitch 2 (BBa_K1054022 ) ===
Various mechanisms about how the aptamer response to inducer have been discovered and we find out a special riboswitch ,which acts differently from K411003.
Various mechanisms about how the aptamer response to inducer have been discovered and we find out a special riboswitch ,which acts differently from K411003.
-
Protocol:
+
<center>
-
# Cultivate the BL21 containing BBa_ in LB at 37℃180rpm until OD600 reaches 0.5.
+
[[File:Static_of_Theo(Helix_Spin).png|500px]]
-
# Induce the bacteria with 1mM IPTG (final concentration), and cultivate them at 37℃180rpm for 4hrs.
+
 
-
# Dilute the suspension culture with LB to 0.1OD600 . Theophylline is added at concentration ranging from 1.00E-06 mol/L to1.00E-01 mol/L.
+
Fig 3.1 Static Performance of Theophylline Riboswitch 2.
-
# Measure the RFU ( ex488/em525) and OD600 synchronously.
+
</center>
-
# Data Analysis
+
<center>
<center>
-
[[File:ZJU-Standardization 3.1.jpg|500px]]
+
[[File:Dynamic_of_Theo_Helix_Spin.png|500px]]
-
Fig 3.1 Graphic Model about how RFU/OD changes at different time and different concentration of Theophylline.
+
Fig 3.2 Graphic Model about how GFP/OD changes at different time and different concentration of Theophylline.
</center>
</center>
<center>
<center>
-
[[File:ZJU-Standardization 3.2.jpg|500px]]
+
[[File:Theo_high2.png|500px]]
-
Fig 3.2 Graphic Model about how GFP Change Rate changes at different time and different concentration of Theophylline.
+
Fig 3.3 Performance Reliability of Theophylline Riboswitch 2 (Cultivated with Theophylline).
 +
</center>
 +
 
 +
<center>
 +
[[File:Theo_low.png|500px]]
 +
 
 +
Fig 3.4 Performance Reliability of Theophylline Riboswitch 2 (Cultivated with NO Theophylline).
</center>
</center>
=== Data Analysis ===
=== Data Analysis ===
-
Theoretically, data processing should consist of two stages including Processing and Calibration, respectively. Lacking enough time and equipment, we cannot finish the Calibration part-which is about to find out the relation between OD600 and the number of cells and also the relation between RFU and mass of GFP-before wiki freeze ,which may have some influence on our results.
+
Theoretically, data processing should consist of two stages including Processing and Calibration, respectively. Lacking enough time and equipment, we cannot finish the Calibration part-which is about to find out the relation about OD600 versus the number of cells and RFU versus mass of GFP-before wiki freeze .
Here comes the Processing Part:
Here comes the Processing Part:
-
(1).Subtract the backgrounds from the raw data
+
'''(1).Subtract the backgrounds from the raw data'''
[[File:ZJU-Standardization-e1.png]]
[[File:ZJU-Standardization-e1.png]]
Line 93: Line 107:
[[File:ZJU-Standardization-e2.png]]
[[File:ZJU-Standardization-e2.png]]
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(2).Calculate the RFU of per unit, P
+
'''(2).Calculate the RFU of per unit, P'''
[[File:ZJU-Standardization-e3.png]]
[[File:ZJU-Standardization-e3.png]]
-
(3).Calculate the whole RFU change rate, S
+
'''(3).Calculate the whole RFU change rate, S'''
[[File:ZJU-Standardization-e4.png]]
[[File:ZJU-Standardization-e4.png]]
-
(4).Plot
+
'''(4).Plot'''
-
If given more time, we may be able to measure the relation between OD and the number of cells AND RFU and mass of GFP, and thus we can calculate the PoPS, which is more accurate and suitable to describe the action of engineered devices.  
+
=== Protocol ===
 +
'''Protocol for Static & Dynamic Performance'''
 +
# Cultivate the BL21 containing riboswitch in LB at 37℃180rpm until OD600 reaches 0.5.
 +
# Induce the bacteria with 1mM IPTG (final concentration), and cultivate them at 37℃180rpm for 4hrs.
 +
# Dilute the suspension culture with LB to 0.1OD600 . Inducer is added at concentration ranging from 1.00E-05 mol/L(for K411003) or 1.00E-06 mol/L(for Atrazine and Theophylline riboswitch 2)to 1.00E-01 mol/L.
 +
# Measure the RFU ( ex488/em525) and OD600 synchronously.
 +
# Data Analysis
 +
 
 +
'''Protocol for Performance Reliability'''
 +
# Cultivate the BL21 containing riboswitch in LB at 37℃180rpm for 15hrs.
 +
# The culture was diluted 1:500 into two identical 5mL cultures. Add inducers to one of the cultures to an input level of 1)5mM(for K511003) 2)1mM(for Atrazine and Theophylline Riboswitch 2).
 +
# The culture was diluted 1:500 in the morning and evening every day for 4 days.
 +
# Each evening, a second culture was inoculated by a 1:500 dilution from the culture for both the high and low input condition. These cultures were grown in the absence of inducers for 8hrs to eliminate the accumulated GFP before assaying performance.
 +
# Samples from both of the second copies were induced with inducers at 37℃180rpm for 45 min. Single-cell fluorescence measurements were carried out on a flow cytometer.
 +
 
 +
Limited by time and resources, it is impossible for us to characterize all riboswitches. Any teams or individuals are warmly welcomed to enrich our riboswitch standardization library.
-
Limited by our time and resources, it is impossible for us to standardize all the riboswitches. Any teams or individuals are warmly welcomed to enrich our riboswitch standardization library.
 
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Latest revision as of 19:47, 28 October 2013

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Characterization: Results

Contents

Characterization of Theophylline Riboswitch (BBa_K411003)

We choose BBa_K411003, a typical riboswitch used by many iGEM teams, to be our first riboswitch to be standardized, which was firstly designed by iGEM10_NYMU-Taipei and has been tested by ZJU_China 2012.

ZJU-Standardization 1.2.jpg

Fig 1.1 shows how the whole GFP Change Rate(GCR) changes at different time and under different concentration. Initially, the culture induced by 0.5M Theophylline has the largest whole GCR. And almost every culture’s GCR experiences decrement along time.


ZJU-Standardization 1.3.jpg

Fig 1.2 GFP Change Rate versus Concentration at 100 minutes after Theophylline was added.


Input compatibility of 411003.png

Fig 1.3 Input Compatibility of K411003

It can be easily observed that the riboswitch’s response to Theophylline is much stronger than to the analogs of theophylline ,including diprophylline, cocaine, caffeine, hypoxanthine and xanthine.

K411003 high.png

Fig 1.4 Performance Reliability of K411003 (Cultivated with Theophylline)

K411003 low2.png

Fig 1.5 Performance Reliability of K411003 (Cultivated with NO Theophylline)

It obvious that the K411003 riboswitch cannot response correctly to theophylline after being cultivated and diluted for 4 days.

Characterization of Atrazine Riboswitch (BBa_K1054008)

Our second choice to be standardized is Atrazine Riboswitch.

Static of Atrazine.png

Fig 2.1 Static Performance of Atrazine Riboswitch

ZJU-Standardization 2.1.jpg

Fig 2.2 Graphic Model about how RFU/OD changes at different time and different concentration of Atrzine.

Input of atrazine.jpg

Fig 2.3 Input Compatibility of Atrazine.

Characterization of Theophylline Riboswitch 2 (BBa_K1054022 )

Various mechanisms about how the aptamer response to inducer have been discovered and we find out a special riboswitch ,which acts differently from K411003.

Static of Theo(Helix Spin).png

Fig 3.1 Static Performance of Theophylline Riboswitch 2.

Dynamic of Theo Helix Spin.png

Fig 3.2 Graphic Model about how GFP/OD changes at different time and different concentration of Theophylline.

Theo high2.png

Fig 3.3 Performance Reliability of Theophylline Riboswitch 2 (Cultivated with Theophylline).

Theo low.png

Fig 3.4 Performance Reliability of Theophylline Riboswitch 2 (Cultivated with NO Theophylline).

Data Analysis

Theoretically, data processing should consist of two stages including Processing and Calibration, respectively. Lacking enough time and equipment, we cannot finish the Calibration part-which is about to find out the relation about OD600 versus the number of cells and RFU versus mass of GFP-before wiki freeze . Here comes the Processing Part:

(1).Subtract the backgrounds from the raw data

ZJU-Standardization-e1.png

ZJU-Standardization-e2.png

(2).Calculate the RFU of per unit, P

ZJU-Standardization-e3.png

(3).Calculate the whole RFU change rate, S

ZJU-Standardization-e4.png

(4).Plot

Protocol

Protocol for Static & Dynamic Performance

  1. Cultivate the BL21 containing riboswitch in LB at 37℃180rpm until OD600 reaches 0.5.
  2. Induce the bacteria with 1mM IPTG (final concentration), and cultivate them at 37℃180rpm for 4hrs.
  3. Dilute the suspension culture with LB to 0.1OD600 . Inducer is added at concentration ranging from 1.00E-05 mol/L(for K411003) or 1.00E-06 mol/L(for Atrazine and Theophylline riboswitch 2)to 1.00E-01 mol/L.
  4. Measure the RFU ( ex488/em525) and OD600 synchronously.
  5. Data Analysis

Protocol for Performance Reliability

  1. Cultivate the BL21 containing riboswitch in LB at 37℃180rpm for 15hrs.
  2. The culture was diluted 1:500 into two identical 5mL cultures. Add inducers to one of the cultures to an input level of 1)5mM(for K511003) 2)1mM(for Atrazine and Theophylline Riboswitch 2).
  3. The culture was diluted 1:500 in the morning and evening every day for 4 days.
  4. Each evening, a second culture was inoculated by a 1:500 dilution from the culture for both the high and low input condition. These cultures were grown in the absence of inducers for 8hrs to eliminate the accumulated GFP before assaying performance.
  5. Samples from both of the second copies were induced with inducers at 37℃180rpm for 45 min. Single-cell fluorescence measurements were carried out on a flow cytometer.

Limited by time and resources, it is impossible for us to characterize all riboswitches. Any teams or individuals are warmly welcomed to enrich our riboswitch standardization library.


Sponsors
The logo of Zhejiang University

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