View source for Part:BBa I13453:British Columbia

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{|
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|width="60px"|''Name'':
|pBAD wild type
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|width="60px"|''Input'':
|width="100px"|[http://openwetware.org/wiki/Arabinose L-arabinose]
|-
|''Output'':
| PoPS 
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[[Part:BBa_I13453|Part Main Page]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
[[Part:BBa_I13453:Design |Part Design]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
[[Part:BBa_I13453:British_Columbia |Characterization]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
[[pBAD Promoter Family |Family]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
[[Part:BBa_I13453:Experience |Add Data]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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All characterization of <partinfo>I13453</partinfo> was done using BW27783 cells carrying the construct <partinfo>K206004</partinfo>. 
==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.
[[Image:PBAD wt - Transfer Function.png|frame|Figure 1. Transfer function of <partinfo>I13453</partinfo>. 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 [[#Summary Datasheet|datasheet]].


==Expression Rate==
We measured the development of RFP over time, in response to intermediate concentrations of arabinose.
[[Image:PBAD wt - 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==
We investigated the specificity of <partinfo>I13453</partinfo> for its ligand, arabinose, by testing it against several other aldopentoses (except rhamnose, a methyl-pentose).
[[Image:PBAD wt - Specificity.png|center|frame|Figure 3. Sugar specificity of <partinfo>I13453</partinfo>. Bars represent individual measurements. Sugars were present at 0.05% w/v except for lyxose which was 0.01% w/v.]]

==Summary Datasheet==

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{|{{Table}}
!Experiment<sup>1</sup>
!Characteristic<sup>1</sup>
!Value<sup>1</sup>
|-
|rowspan="2"|[[#Transfer Function|'''Transfer Function''']]
|''Hill coefficient''
|2.19
|-
|''Switch Point''
|4.08E-6 M L-arabinose, exogenous
|-
|[[#Expression Rate|'''Rate of Expression''']]
|''Response Time''
|~10 hours
|-
|rowspan="2"|[[#Specificity|'''Input compatibility''']]
|''Strong response to''
|L-arabinose
|-
|''No response to''
|Ribose, xylose, lyxose, rhamnose
|-
|}
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<sup>1</sup>Measured by the UBC iGEM Team 2009

==General Characterization Protocols==
# Inoculate 5mL LB-Chlor medium in a sterile test tube with a colony from a streak plate of pSB1C3-K206004-transformed BW27783 cells.
# Grow overnight at 37C, shaking at 210rpm.
# Measure OD600 and dilute to OD600=0.25A in 15mL of LB-Chlor with added arabinose (or other sugars).
# Grow at 37C, shaking at 210rpm.
# 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).
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