Difference between revisions of "Part:BBa J23117:Experience"

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<I>iGEM Team Göttingen 2013</I>
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Additionally to our characterization of this part, we also used it for our reporter system and it worked very good! We also improved it by switching the pre- and suffix, basically inverting it. This way, we were able to use it in an "inverted" expression unit on the same vector as our reporter system. For further information see: https://parts.igem.org/wiki/index.php?title=Part:BBa_K1045011
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<I>iGEM15_UNITN-Trento</I>
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We used the part BBa_J23117 in the InterLab Measurement context. We assembled a measurement device by inserting the part <html><a href="https://parts.igem.org/Part:BBa_I13504">BBa_I13504</a></html> amplified by PCR into the part BBa_J23117. The device was characterized in <i>E. coli</i> NEB10&beta;, JM109, and NEB Express by measuring GFP expression with a cuvette-based spectrofluorometer, a fluorescence plate reader, and a flow cytometer. We also confirmed the promoter activity with a cell-free S30 extract system and measured mRNA by RT-qPCR. <br> <br>
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For a better understanding on protocols and characterizations, please check out our Wiki page <html><a href="http://2015.igem.org/Team:UNITN-Trento/Measurement">UNITN-Trento iGEM 2015</a>! <br> <br></html>
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====Evaluation of Anderson promoter J23117 in ''E. coli'' by [http://2013.igem.org/Team:Goettingen iGEM Göttingen 2013]====
 
====Evaluation of Anderson promoter J23117 in ''E. coli'' by [http://2013.igem.org/Team:Goettingen iGEM Göttingen 2013]====
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Promoter 2:[[Part:BBa_J23116|BBa_J23116]] <br />
 
Promoter 2:[[Part:BBa_J23116|BBa_J23116]] <br />
 
Promoter 3:[[Part:BBa_J23110|BBa_J23110]] <br />
 
Promoter 3:[[Part:BBa_J23110|BBa_J23110]] <br />
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Promoter 4:[[Part:BBa_J23118|BBa_J23118]]
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Promoter 4:[[Part:BBa_J23118|BBa_J23118]] <br />
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The promoter strength was measured by using the reporter gene ''rfp''.<br />
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Three different approaches were used: '''1. RFP measurement''', '''2. qRT-PCR analyses''' and '''3. single cell microscopy'''. Moreover, the first and the second approach characterised the promoter activity along the growth curve and to three important time points, respectively.<br />
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Taken together, the majority of our results from these approaches showed that [[Part:BBa_J23117|BBa_J23117]] has the lowest promoter activity compared to [[Part:BBa_J23116|BBa_J23116]], [[Part:BBa_J23110|BBa_J23110]] and [[Part:BBa_J23118|BBa_J23118]]. For more details, visit the discussion on our wikipage.
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[[File:J23117.jpg|420px|thumb|left|'''Fig. 1.''' RFP and qRT-PCR promoter analyses.]]
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[[File:promoterGoe.jpg|420px|thumb|right|'''Fig. 2.''' Microscopic promoter analyses on single cell level <br />
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on the left side: bright field (BR), on the right side: RFP filter (rfp) <br />
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P1: [[Part:BBa_J23117|BBa_J23117]], P2: [[Part:BBa_J23116|BBa_J23116]], P3: [[Part:BBa_J23110|BBa_J23110]], P4: [[Part:BBa_J23118|BBa_J23118]], P8: control ]]
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<I>University of Texas at Austin iGEM 2019</I>
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<h3>UT Austin iGEM 2019: Characterization of metabolic burden of the Anderson Series</h3>
  
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<h4>Description</h4>
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[[File:promoterGoe.jpg|400|left]]
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The 2019 UT Austin iGEM team transformed the Anderson Series promoters into our 'burden monitor' DH10B strain of E. coli, which contains a constitutive GFP cassette in the genome of the cell. GFP expression fluctuates depending on the number of ribosomes available. Using this strain, we characterized the relative burden (percent reduction in growth rate) of each Anderson Series part. Our results showed a range of growth rate reductions for each of these parts due to ribosomal reallocation from the genome of the host cell, towards the expression of RFP. Anderson Series parts with strong promoters are depicted with darker red colors and Anderson Series parts with weak promoters are depicted with lighter pink colors to show relative RFP expression.
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We saw a positive correlation between relative promoter strength and metabolic burden; parts with stronger promoters expressed less GFP and had a lower growth rate than parts with weaker promoters. The regression line for the graph below was constructed by measuring the burden of 5 parts that were created by the 2019 UT Austin iGEM team that each contained an Anderson Series promoter (<partinfo>J23104</partinfo> or <partinfo>J23110</partinfo>), an RBS of varying strength, and a BFP reporter. For more information on characterization of these parts through the burden monitor, visit our team’s wiki page: [https://https://2019.igem.org/Team:Austin_UTexas]
  
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<img src = "https://static.igem.org/mediawiki/parts/a/a0/AndersonCharacterization.jpg" style = "width:550px;height:500px">
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<figcaption><b>Fig.1:</b>Growth vs GFP Expression graph showing the relative burden positions of the Anderson Series promoters. The parts with strong promoters are depicted in dark red and are clustered near the bottom of the graph because they have lower growth rates and express lower levels of GFP as a result of high cellular burden. The parts with weaker promoter are depicted in light pink ad are clustered near the top of the graph because they have higher growth rates and express higher levels of GFP as a result of low cellular burden.</figcaption>
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<img src = "https://static.igem.org/mediawiki/parts/8/80/T--Austin_Utexas--andersontable.png" style = "width:545px;height:375px">
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<figcaption><b>Table.1:</b> Burden measurements for the Anderson Series promoters measured as percent reduction in growth rate ± 95% confidence interval. </figcaption>
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<h4>Importance of Characterizing Burden</h4>
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<p> Although often we cannot avoid using a specific burdensome part, knowing in advance that it is burdensome, and that it has a high chance of mutating into a non-functional genetic device, can help with troubleshooting and coming up with alternatives. In the specific case of fluorescent protein-expressing devices, Fluorescence-activated cell sorting (FACS) can be used to filter out individual cells that meet a certain fluorescence threshold. This way, the cells expressing lower levels of the fluorescent protein are weeded out of the population.</p>

Latest revision as of 20:18, 21 October 2019

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_J23117

Evaluation of Anderson promoter J23117 in B. subtilis by iGEM-Team LMU-Munich 2012

This Anderson promoter was evaluated without fused RFP with the lux operon as a reporter in B. subtilis. See the new BioBrick BBa_K823013 without RFP and have a look at the [http://2012.igem.org/Team:LMU-Munich/Data/Anderson Data] from the evaluation in B. subtilis.

User Reviews

UNIQ3a6c4cc60ef7f215-partinfo-00000000-QINU UNIQ3a6c4cc60ef7f215-partinfo-00000001-QINU

•••••

iGEM Team Göttingen 2013

Additionally to our characterization of this part, we also used it for our reporter system and it worked very good! We also improved it by switching the pre- and suffix, basically inverting it. This way, we were able to use it in an "inverted" expression unit on the same vector as our reporter system. For further information see: https://parts.igem.org/wiki/index.php?title=Part:BBa_K1045011


UNIQ3a6c4cc60ef7f215-partinfo-00000003-QINU

•••••

iGEM15_UNITN-Trento

We used the part BBa_J23117 in the InterLab Measurement context. We assembled a measurement device by inserting the part BBa_I13504 amplified by PCR into the part BBa_J23117. The device was characterized in E. coli NEB10β, JM109, and NEB Express by measuring GFP expression with a cuvette-based spectrofluorometer, a fluorescence plate reader, and a flow cytometer. We also confirmed the promoter activity with a cell-free S30 extract system and measured mRNA by RT-qPCR.

For a better understanding on protocols and characterizations, please check out our Wiki page UNITN-Trento iGEM 2015!


Evaluation of Anderson promoter J23117 in E. coli by [http://2013.igem.org/Team:Goettingen iGEM Göttingen 2013]



Shown here:
Upper two pictures: Growth curves of promoter strains on the left, growth curves of control strains on the right. Three biological replicates are shown.
Middle two pictures: RFP/OD600 of promoter strains on the left, RFP/OD600 of control strains on the right. Three biological replicates are shown.
Bottom three pictures: qRT PCR promoter analyses in three different growth phases. Promoters are normalised against BBa_J23117 .

Promotor 1:BBa_J23117
Promoter 2:BBa_J23116
Promoter 3:BBa_J23110
Promoter 4:BBa_J23118


The promoter strength was measured by using the reporter gene rfp.
Three different approaches were used: 1. RFP measurement, 2. qRT-PCR analyses and 3. single cell microscopy. Moreover, the first and the second approach characterised the promoter activity along the growth curve and to three important time points, respectively.
Taken together, the majority of our results from these approaches showed that BBa_J23117 has the lowest promoter activity compared to BBa_J23116, BBa_J23110 and BBa_J23118. For more details, visit the discussion on our wikipage.


Fig. 1. RFP and qRT-PCR promoter analyses.


Fig. 2. Microscopic promoter analyses on single cell level
on the left side: bright field (BR), on the right side: RFP filter (rfp)
P1: BBa_J23117, P2: BBa_J23116, P3: BBa_J23110, P4: BBa_J23118, P8: control


•••••

University of Texas at Austin iGEM 2019

UT Austin iGEM 2019: Characterization of metabolic burden of the Anderson Series

Description

The 2019 UT Austin iGEM team transformed the Anderson Series promoters into our 'burden monitor' DH10B strain of E. coli, which contains a constitutive GFP cassette in the genome of the cell. GFP expression fluctuates depending on the number of ribosomes available. Using this strain, we characterized the relative burden (percent reduction in growth rate) of each Anderson Series part. Our results showed a range of growth rate reductions for each of these parts due to ribosomal reallocation from the genome of the host cell, towards the expression of RFP. Anderson Series parts with strong promoters are depicted with darker red colors and Anderson Series parts with weak promoters are depicted with lighter pink colors to show relative RFP expression. We saw a positive correlation between relative promoter strength and metabolic burden; parts with stronger promoters expressed less GFP and had a lower growth rate than parts with weaker promoters. The regression line for the graph below was constructed by measuring the burden of 5 parts that were created by the 2019 UT Austin iGEM team that each contained an Anderson Series promoter (BBa_J23104 or BBa_J23110), an RBS of varying strength, and a BFP reporter. For more information on characterization of these parts through the burden monitor, visit our team’s wiki page: [1]

Fig.1:Growth vs GFP Expression graph showing the relative burden positions of the Anderson Series promoters. The parts with strong promoters are depicted in dark red and are clustered near the bottom of the graph because they have lower growth rates and express lower levels of GFP as a result of high cellular burden. The parts with weaker promoter are depicted in light pink ad are clustered near the top of the graph because they have higher growth rates and express higher levels of GFP as a result of low cellular burden.


Table.1: Burden measurements for the Anderson Series promoters measured as percent reduction in growth rate ± 95% confidence interval.

Importance of Characterizing Burden

Although often we cannot avoid using a specific burdensome part, knowing in advance that it is burdensome, and that it has a high chance of mutating into a non-functional genetic device, can help with troubleshooting and coming up with alternatives. In the specific case of fluorescent protein-expressing devices, Fluorescence-activated cell sorting (FACS) can be used to filter out individual cells that meet a certain fluorescence threshold. This way, the cells expressing lower levels of the fluorescent protein are weeded out of the population.