Difference between revisions of "Part:BBa J23103:Experience"

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<I>University of Texas at Austin iGEM 2019</I>
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====Characterization of the Anderson series through burden monitoring by the University of Texas at Austin's 2019 iGEM team====
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===Description===
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Our team transformed the Anderson series of RFP reporters (J23101, J23113, J23104, J23107, J23117 in pSBC13 backbone) into our constitutive GFP burden monitor <I>E. coli</I> strain and measured the relative burden of each part. They contained a constitutive GFP sequence in the genome which serves as a way to measure the constructs’ ribosome allocation. Our results show a certain reduction ingrowth rate for each part as a result of ribosome misallocation away from the genome and towards the plasmid containing the construct. The promoter strengths associated with each RFP reporter construct shows that parts with stronger promoters express less GFP and have a reduced growth rate when compared to the constructs containing weaker promoters. The promoters associated with these RFP reporters were used to create a series of BFP reporters, also transformed into our GFP burden monitor strain, and create the regression on the figure below. For more information on characterization of these parts through burden monitoring and evolutionary stability experiments, visit our team’s wiki page: [https://https://2019.igem.org/Team:Austin_UTexas]
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[[Image: AndersonCharacterization.jpg|450px]]

Revision as of 04:10, 10 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_J23103

Use of this promoter by team Glasgow 2014

We found this promoter is actually identical in sequence to BBa_J23112

BBa_J23116, BBa_J23106, BBa_J23103, and BBa_J23112 were used to express motA and motB together in our composite biobricks:
BBa_ K1463773, BBa_ K1463772, BBa_ K1463770, and BBa_ K1463771 respectively.
These composite biobricks were used to complement the swimming defect of a motA E. coli mutant.
We found that swimming was restored in the following order:
BBa_J23116 > BBa_J23106 > BBa_J23103 = BBa_J23112.
Examination of the sequences of BBa_J23103 and BBa_J23112 showed that they are identical, despite showing different levels of RFP expression in their initial characterisation!

>BBa_J23103 Part-only sequence (35 bp) ctgatagctagctcagtcctagggattatgctagc

>BBa_J23112 Part-only sequence (35 bp) ctgatagctagctcagtcctagggattatgctagc

Evaluation of Anderson promoter J23103 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_K823007 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

UNIQ6bbb7e26d50e7005-partinfo-00000000-QINU

•••••

iGEM HKU 2011

To start characterizing the promoters, we have performed the red florescence intensity measurements for our selected plasmid in the E.Coli MG1655 strain. The data collected is shown below. It is found that promoter J23106 can lead to a higher expression since the fluorescence intensity per OD600 is the highest, while J23103, J23109, J23116 have relative low expression and fluorescence. As our selected promoters have different strength, thus our team is able to use them to fine tune the protein expression.

mRFP fluorescence intensity under different promoters

UNIQ6bbb7e26d50e7005-partinfo-00000002-QINU


•••••

University of Texas at Austin iGEM 2019

Characterization of the Anderson series through burden monitoring by the University of Texas at Austin's 2019 iGEM team

Description

Our team transformed the Anderson series of RFP reporters (J23101, J23113, J23104, J23107, J23117 in pSBC13 backbone) into our constitutive GFP burden monitor E. coli strain and measured the relative burden of each part. They contained a constitutive GFP sequence in the genome which serves as a way to measure the constructs’ ribosome allocation. Our results show a certain reduction ingrowth rate for each part as a result of ribosome misallocation away from the genome and towards the plasmid containing the construct. The promoter strengths associated with each RFP reporter construct shows that parts with stronger promoters express less GFP and have a reduced growth rate when compared to the constructs containing weaker promoters. The promoters associated with these RFP reporters were used to create a series of BFP reporters, also transformed into our GFP burden monitor strain, and create the regression on the figure below. For more information on characterization of these parts through burden monitoring and evolutionary stability experiments, visit our team’s wiki page: [1]

AndersonCharacterization.jpg