Difference between revisions of "Part:BBa K1104244:Experience"
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The testing result of this device can be viewed in the following link:(br)
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The testing result of this device can be viewed in the following link:<br>
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(https://parts.igem.org/Part:BBa_K1104204:Experience Part:BBa_K1104204 Experience])
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([https://parts.igem.org/Part:BBa_K1104204:Experience BBa_K1104204 Experience])
  
  
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===Characterization===
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<partinfo>BBa_K1104244 AddReview 5</partinfo>
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<I>iGEM2019[http://2019.igem.org/Team:BIT# BIT]</I>
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iGEM2019 [http://2019.igem.org/Team:BIT# BIT]
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Expression of <bbpart>BBa-K1104244</bbpart> in trans5α strain and induction of hydrogen peroxide
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<partinfo>BBa_K1104244 AddReview 5</partinfo>
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We introduced <bbpart>BBa--K1104244</bbpart> into tran5α competent cells, applied them to LB plates containing 300 μg/mL chloramphenicol, inverted overnight at 37 ° C, and then picked monoclonal colonies up into chloramphenicol-resistant LB solid medium overnight.  Then take 400 μL of bacterial solution inoculated in LB medium at 37 ° C 130 r / min shaker shaking culture for 2-3 hours, when their OD600=0.2-0.4(Logarithmic period), adding different concentrations of hydrogen peroxide, induced at 37 ° C 130r / min for 2 - 3 hours to generate GFP.
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We conducted two sets of experiments, and set different concentration gradients to observe the induced expression of GFP.
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[[File:BIT 2019 DNA expression of BB-1104244 1.png |460px]]
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[[File:BIT 2019 DNA expression of BB-1104244 2.png |460px]]
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After being induced at 37 ° C 130r / min for 2 .5 hours, add 200 μL bacterial solution into a black 96-well plate (Corning, USA) for fluorescence determination. We set the fluorescence excitation wavelength to 488 nm, and the emission wavelength to 516nm, and the OD600 was measured at the same time.The two sets of data were obtained by scanning at room temperature in a multi-function microplate reader.
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The data obtained is as follows:
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First set:
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[[File:BIT 2019 DNA expression of BB-1104244 3.png |460px]]
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The promoter strengths are standardized by setting AhpCp2d1 in the absence of H202 as 1
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[[File:BIT 2019 DNA expression of BB-1104244 4.png |460px]]
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specific fluorescence units：SFU=RFU/OD600
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Second set:
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[[File:BIT 2019 DNA expression of BB-1104244 5.png |460px]]
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The promoter strengths are standardized by setting AhpCp2d1 in the absence of H202 as 1
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[[File:BIT 2019 DNA expression of BB-1104244 6.png |460px]]
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specific fluorescence units：SFU=RFU/OD600
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It can be seen from the above experiment that within a certain range, as the concentration of hydrogen peroxide increases, the amount of fluorescent expression induced by the promoter-induced expression increases. This is basically consistent with the results obtained by the iGEM13_NYMU-Taipei (2013-09-16) team.

Latest revision as of 09:49, 21 October 2019

The testing result of this device can be viewed in the following link:
(BBa_K1104204 Experience)


Applications of BBa_K1104244

User Reviews

UNIQd069f0c6812be467-partinfo-00000000-QINU UNIQd069f0c6812be467-partinfo-00000001-QINU


Characterization

•••••

iGEM2019[http://2019.igem.org/Team:BIT# BIT]

iGEM2019 [http://2019.igem.org/Team:BIT# BIT] Expression of BBa-K1104244 in trans5α strain and induction of hydrogen peroxide

•••••

We introduced BBa--K1104244 into tran5α competent cells, applied them to LB plates containing 300 μg/mL chloramphenicol, inverted overnight at 37 ° C, and then picked monoclonal colonies up into chloramphenicol-resistant LB solid medium overnight. Then take 400 μL of bacterial solution inoculated in LB medium at 37 ° C 130 r / min shaker shaking culture for 2-3 hours, when their OD600=0.2-0.4(Logarithmic period), adding different concentrations of hydrogen peroxide, induced at 37 ° C 130r / min for 2 - 3 hours to generate GFP. We conducted two sets of experiments, and set different concentration gradients to observe the induced expression of GFP.

BIT 2019 DNA expression of BB-1104244 1.png

BIT 2019 DNA expression of BB-1104244 2.png

After being induced at 37 ° C 130r / min for 2 .5 hours, add 200 μL bacterial solution into a black 96-well plate (Corning, USA) for fluorescence determination. We set the fluorescence excitation wavelength to 488 nm, and the emission wavelength to 516nm, and the OD600 was measured at the same time.The two sets of data were obtained by scanning at room temperature in a multi-function microplate reader. The data obtained is as follows:

First set:

BIT 2019 DNA expression of BB-1104244 3.png

The promoter strengths are standardized by setting AhpCp2d1 in the absence of H202 as 1

BIT 2019 DNA expression of BB-1104244 4.png

specific fluorescence units：SFU=RFU/OD600

Second set:

BIT 2019 DNA expression of BB-1104244 5.png

The promoter strengths are standardized by setting AhpCp2d1 in the absence of H202 as 1

BIT 2019 DNA expression of BB-1104244 6.png

specific fluorescence units：SFU=RFU/OD600


It can be seen from the above experiment that within a certain range, as the concentration of hydrogen peroxide increases, the amount of fluorescent expression induced by the promoter-induced expression increases. This is basically consistent with the results obtained by the iGEM13_NYMU-Taipei (2013-09-16) team.