Difference between revisions of "Part:BBa K389015:Experience"

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===Applications of BBa_K389015===
 
===Applications of BBa_K389015===
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====Transfer function====
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The data for the transfer function was measured and analyzed as described below. The data was fitted with a dose response function of the form
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[[Image:Bielefeld_Doseresponse_fit.jpg|175px|center]] <div align="right">(1)</div>
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with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x<sub>0</sub>). Figure 1 shows the measured normalized specific production rates q<sub>P,n</sub> plotted against the logarithm of the concentration of the inductor acetosyringone in µM. The fit has an R<sup>2</sup> = 0.98.
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[[Image:Bielefeld_Fit_Luc_final.jpg|600px|thumb|center|'''Fig. 1: Transfer function for the part <partinfo>K389015</partinfo> (R<sup>2</sup> = 0.98).''']]
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The important data from the transfer function is summarized in table 1:
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<center>
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Table 1: Data from the transfer function for the part <partinfo>K389015</partinfo>.
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{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"
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|-
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!style="border-style: solid; border-width: 1px"| Parameter
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!style="border-style: solid; border-width: 1px"| Value
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|-
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|style="border-style: solid; border-width: 1px"| Hill coefficient
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|style="border-style: solid; border-width: 1px"| 1.092
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|-
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|style="border-style: solid; border-width: 1px"| [https://parts.igem.org/Switch_Point Switch point]
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|style="border-style: solid; border-width: 1px"| 31.6 µM
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|style="border-style: solid; border-width: 1px"| Top asymptote
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|style="border-style: solid; border-width: 1px"| 2.16
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|}
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</center>
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The fully induced VirA/G signaling system with luciferase read-out has a 2.2 fold increased expression compared to the uninduced system. The Hill coefficient is > 1, so a positive cooperation can be observed ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 D Chu ''et al.'', 2009]). The [https://parts.igem.org/Switch_Point switch point] of the system is at about 32 µM, so this is the concentration at which the device output is 50% of the maximum output.
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====Response time====
 
====Response time====
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The system needs at least one hour to show a measurable reaction to an induction with acetosyringone. In the following illustration the reaction of the system to induction with 200 µM acetosyringone in the exponential growth phase is shown. For a good separation of the induced system from the uninduced system at least two hours are needed.  
 
The system needs at least one hour to show a measurable reaction to an induction with acetosyringone. In the following illustration the reaction of the system to induction with 200 µM acetosyringone in the exponential growth phase is shown. For a good separation of the induced system from the uninduced system at least two hours are needed.  
  
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[[Image:Bielefeld_Induktionsverhalten_luc.jpg|600px|center|thumb|Cultivation of <partinfo>K389015</partinfo> in ''Escherichia coli'' DB3.1. Induction with 200 µM acetosyringone during the exponential growth phase.]]
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[[Image:Bielefeld_Induktionsverhalten_luc.jpg|600px|center|thumb|'''Fig. 2: Cultivation of <partinfo>K389015</partinfo> in ''Escherichia coli'' DB3.1. Induction with 200 µM acetosyringone during the exponential growth phase.''']]
  
  
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[[Image:Bielefeld_Quotient_RLU.jpg|200px|center]] <div align="right">(1)</div>
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[[Image:Bielefeld_Quotient_RLU.jpg|200px|center]] <div align="right">(2)</div>
  
  
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As seen above, at least one hour is needed to separate the induced luminescence signal from the uninduced, so ɸ<sub>I</sub> > 1. Within a cultivation ɸ<sub>I</sub> is rising during the first hours and is decreasing after it reached a maximum at OD<sub>600</sub> ~ 1. This is shown in the following figure:  
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As seen above, at least one hour is needed to separate the induced luminescence signal from the uninduced, so ɸ<sub>I</sub> > 1. Within a cultivation ɸ<sub>I</sub> is rising during the first hours and is decreasing after it reached a maximum at OD<sub>600</sub> ~ 1. This is shown in figure 3:  
  
  

Revision as of 17:39, 25 October 2010

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Applications of BBa_K389015

Transfer function

The data for the transfer function was measured and analyzed as described below. The data was fitted with a dose response function of the form


Bielefeld Doseresponse fit.jpg
(1)


with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x0). Figure 1 shows the measured normalized specific production rates qP,n plotted against the logarithm of the concentration of the inductor acetosyringone in µM. The fit has an R2 = 0.98.


Error creating thumbnail: Invalid thumbnail parameters
Fig. 1: Transfer function for the part BBa_K389015 (R2 = 0.98).


The important data from the transfer function is summarized in table 1:

Table 1: Data from the transfer function for the part BBa_K389015.

Parameter Value
Hill coefficient 1.092
Switch point 31.6 µM
Top asymptote 2.16


The fully induced VirA/G signaling system with luciferase read-out has a 2.2 fold increased expression compared to the uninduced system. The Hill coefficient is > 1, so a positive cooperation can be observed ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 D Chu et al., 2009]). The switch point of the system is at about 32 µM, so this is the concentration at which the device output is 50% of the maximum output.


Response time

The system needs at least one hour to show a measurable reaction to an induction with acetosyringone. In the following illustration the reaction of the system to induction with 200 µM acetosyringone in the exponential growth phase is shown. For a good separation of the induced system from the uninduced system at least two hours are needed.

Error creating thumbnail: Invalid thumbnail parameters
Fig. 2: Cultivation of BBa_K389015 in Escherichia coli DB3.1. Induction with 200 µM acetosyringone during the exponential growth phase.


Data Analysis

Because the luciferase accumulation is very different in different cultivations, the uninduced negative control was used as internal standard. To show the behaviour of the VirA/G signaling system when induced, the ratio ɸI between induced (i) and uninduced (u) relative luminescence unit (RLU) per OD600 is calculated:


Bielefeld Quotient RLU.jpg
(2)


As seen above, at least one hour is needed to separate the induced luminescence signal from the uninduced, so ɸI > 1. Within a cultivation ɸI is rising during the first hours and is decreasing after it reached a maximum at OD600 ~ 1. This is shown in figure 3:


To measure the ratio of increasing promoteractivity by inducing the system ɸI samples for analyzation should be taken at OD600 ~ 1.

Protocols

1. bla


User Reviews

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