Difference between revisions of "Part:BBa K206000:Characterization"
 
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=Characterization=
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__NOTOC__
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{|
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|<div style="align: center; valign: center; font-family: Arial; font-size: 12pt">
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{|
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|-
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|width="60px"|''Name'':
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|pBAD strong
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|-
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|width="60px"|''Input'':
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|width="100px"|[http://openwetware.org/wiki/Arabinose L-arabinose]
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|-
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|''Output'':
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| PoPS
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|}
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</div>
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<hr width="680px">
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<div class="noprint" style="padding: 10px; color: #ffffff; background-color: #C0C0C0; width: 680px; align: center">
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<center>
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[[Part:BBa_K206000 |Part Main Page]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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[[Part:BBa_K206000:Design |Part Design]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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[[Part:BBa_K206000:Characterization |Characterization]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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[[pBAD Promoter Family |Family]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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[[Part:BBa_K206000:Experience |Add Data]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</center>
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</div>
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<div style="padding: 00px; width: 680px; border: 0px solid #000000;">
 
All characterization of <partinfo>K206000</partinfo> was done using BW27783 cells carrying the construct <partinfo>K206002</partinfo>.  
 
All characterization of <partinfo>K206000</partinfo> was done using BW27783 cells carrying the construct <partinfo>K206002</partinfo>.  
 
==Transfer Function==
 
==Transfer Function==
 
We attempted to find the transfer function relating input concentration of arabinose to PoPS by measuring RFP production in response to varying arabinose concentration.
 
We attempted to find the transfer function relating input concentration of arabinose to PoPS by measuring RFP production in response to varying arabinose concentration.
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[[Image:PBAD strong - Transfer Function.png|center|frame|Figure 1. Transfer function of <partinfo>K206000</partinfo>. Points represent individual measurements. The line is of a Hill equation fitted to our data.]]
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We fit a Hill equation to the data from our experiment and used it to determine the values listed in our [[#Summary Datasheet|datasheet]].
  
  
 
==Expression Rate==
 
==Expression Rate==
 
We measured the development of RFP over time, in response to intermediate concentrations of arabinose.
 
We measured the development of RFP over time, in response to intermediate concentrations of arabinose.
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[[Image:PBAD strong - Timecourse.png|center|frame|Figure 2. Fluorescence development after induction with arabinose. Points represent means of 3 separate measurements. Error bars represent the standard deviation. Control measurements were of BW27783 cells lacking the construct.]]
  
 
==Specificity==
 
==Specificity==
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We investigated the specificity of <partinfo>K206000</partinfo> for its ligand, arabinose, by testing it against several other aldopentoses of similar structure.
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We investigated the specificity of <partinfo>K206000</partinfo> for its ligand, arabinose, by testing it against several other aldopentoses (except rhamnose, a methyl-pentose).
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[[Image:PBAD strong - Specificity.png|center|frame|Figure 3. Sugar specificity of <partinfo>K206000</partinfo>. Bars represent individual measurements. Sugars were present at 0.05% w/v except for lyxose which was 0.01% w/v.]]
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==Summary Datasheet==
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<center>
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{|{{Table}}
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!Experiment<sup>1</sup>
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!Characteristic<sup>1</sup>
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!Value<sup>1</sup>
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|-
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|rowspan="2"|[[#Transfer Function|'''Transfer Function''']]
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|''Hill coefficient''
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|2.26
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|-
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|''Switch Point''
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|3.14E-6 M L-arabinose, exogenous
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|-
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|[[#Expression Rate|'''Rate of Expression''']]
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|''Response Time''
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|~10 hours
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|-
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|rowspan="2"|[[#Specificity|'''Input compatibility''']]
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|''Strong response to''
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|L-arabinose
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|-
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|''No response to''
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|Ribose, xylose, lyxose, rhamnose
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|-
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|}
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</center>
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<sup>1</sup>Measured by the UBC iGEM Team 2009
  
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===General Characterization Protocols===
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==General Characterization Protocols==
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1. Inoculate 5mL LB-Chlor medium in a sterile test tube with a colony from a streak plate of pSB1C3-K206000-transformed BW27783 cells.
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# Inoculate 5mL LB-Chlor medium in a sterile test tube with a colony from a streak plate of pSB1C3-K206002-transformed BW27783 cells.
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2. Grow overnight at 37C, shaking at 210rpm.
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# Grow overnight at 37C, shaking at 210rpm.
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3. Measure OD600 and dilute to OD600=0.25A in 15mL of LB-Chlor with added arabinose (or other sugars).
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# Measure OD600 and dilute to OD600=0.25A in 15mL of LB-Chlor with added arabinose (or other sugars).
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4. Grow at 37C, shaking at 210rpm.
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# Grow at 37C, shaking at 210rpm.
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5. At appropriate timepoints (~24 hours for Transfer Function and Specificity experiments), remove samples for measuring OD600 (1/10 dilution) and fluorescence (fluorescence samples were frozen at -80C as cell pellets and measured together).
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# At appropriate timepoints (~24 hours for Transfer Function and Specificity experiments), remove samples for measuring OD600 (1/10 dilution) and fluorescence (fluorescence samples were frozen at -80C as cell pellets and measured together).
  
 
Sugars were made up to 10% w/v or 500uM stock solutions in water and filter sterilized with a 0.45um filter.
 
Sugars were made up to 10% w/v or 500uM stock solutions in water and filter sterilized with a 0.45um filter.
 
RFP fluorescence was measured by pelleting 500uL of cells and resuspending in PBS, then collecting 50000 events on a Becton-Dickinson Influx flow cytometer. Fluorescence was calculated by finding the mean of the RFP histogram for each sample, normalized to OD600 (when possible).
 
RFP fluorescence was measured by pelleting 500uL of cells and resuspending in PBS, then collecting 50000 events on a Becton-Dickinson Influx flow cytometer. Fluorescence was calculated by finding the mean of the RFP histogram for each sample, normalized to OD600 (when possible).
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</div>

Latest revision as of 03:25, 22 October 2009


Name: pBAD strong
Input: [http://openwetware.org/wiki/Arabinose L-arabinose]
Output: PoPS

Part Main Page        Part Design        Characterization        Family        Add Data       

All characterization of BBa_K206000 was done using BW27783 cells carrying the construct BBa_K206002.

Transfer Function

We attempted to find the transfer function relating input concentration of arabinose to PoPS by measuring RFP production in response to varying arabinose concentration.

Figure 1. Transfer function of BBa_K206000. Points represent individual measurements. The line is of a Hill equation fitted to our data.

We fit a Hill equation to the data from our experiment and used it to determine the values listed in our datasheet.


Expression Rate

We measured the development of RFP over time, in response to intermediate concentrations of arabinose.

Figure 2. Fluorescence development after induction with arabinose. Points represent means of 3 separate measurements. Error bars represent the standard deviation. Control measurements were of BW27783 cells lacking the construct.

Specificity

We investigated the specificity of BBa_K206000 for its ligand, arabinose, by testing it against several other aldopentoses (except rhamnose, a methyl-pentose).

Figure 3. Sugar specificity of BBa_K206000. Bars represent individual measurements. Sugars were present at 0.05% w/v except for lyxose which was 0.01% w/v.

Summary Datasheet

Experiment1 Characteristic1 Value1
Transfer Function Hill coefficient 2.26
Switch Point 3.14E-6 M L-arabinose, exogenous
Rate of Expression Response Time ~10 hours
Input compatibility Strong response to L-arabinose
No response to Ribose, xylose, lyxose, rhamnose

1Measured by the UBC iGEM Team 2009

General Characterization Protocols

  1. Inoculate 5mL LB-Chlor medium in a sterile test tube with a colony from a streak plate of pSB1C3-K206002-transformed BW27783 cells.
  2. Grow overnight at 37C, shaking at 210rpm.
  3. Measure OD600 and dilute to OD600=0.25A in 15mL of LB-Chlor with added arabinose (or other sugars).
  4. Grow at 37C, shaking at 210rpm.
  5. At appropriate timepoints (~24 hours for Transfer Function and Specificity experiments), remove samples for measuring OD600 (1/10 dilution) and fluorescence (fluorescence samples were frozen at -80C as cell pellets and measured together).

Sugars were made up to 10% w/v or 500uM stock solutions in water and filter sterilized with a 0.45um filter. RFP fluorescence was measured by pelleting 500uL of cells and resuspending in PBS, then collecting 50000 events on a Becton-Dickinson Influx flow cytometer. Fluorescence was calculated by finding the mean of the RFP histogram for each sample, normalized to OD600 (when possible).