Difference between revisions of "Part:BBa J23109:Experience"

(→‎Description)
(→‎Characterization experiment by qrtPCR on BBa_J23100, BBa_J23104, BBa_J23105, BBa_J23106, BBa_J23109, BBa_J23112, BBa_J23113, BBa_J23114 by iGEM Team Göttingen (by C. Krüger and J. Kampf))
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=====Description=====
 
=====Description=====
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We used quantitative real time PCR to examine the expression rate of eight different constitutive promoter constructs of from the parts registry. The reported activities of these promoters are given as the relative fluorescence of these plasmids in strain TG1 ([http://2012.igem.org/Team:Goettingen/Project/Methods#Quantitative_Real-Time_PCR parts registry: part:BBa_J23100]). Promoter constructs were cloned into the vector pSB1C3 and expressed in <i>E.coli BL21DE3</i> grown in LB-media (lysogeny broth). The measurements were performed for each construct and reference as a triplet. Additionally, we concluded H<sub>2</sub>O as negative control to predict possible contamination. For the evaluation of our results,  the 2–ΔΔCT (Livak) Method was applied.  
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We used quantitative real-time PCR as a powerful tool for quantitation of gene expression. We used this method to examine the expression rate of eight different constitutive promoter constructs from the [https://parts.igem.org/Promoters/Catalog/Anderson Anderson family] of the parts registry.  
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The data were compared to the determined expression rates from the part registry in the following figure.  
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You can find detailed information for our qrtPCR approach [http://2012.igem.org/Team:Goettingen/Project/Methods#-.3E_Experimental_design here].<br>
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The reported activities of these promoters are given as the relative fluorescence of these plasmids in strain TG1 [https://parts.igem.org/Promoters/Catalog/Anderson]. Promoter constructs were cloned into the vector pSB1C3 and expressed in <i>E.coli</i> BL21DE3 grown in LB-media (lysogeny broth). The measurements were performed for each construct and reference as a triplet. Additionally, we included H<sub>2</sub>O as negative control to predict possible contamination. For the evaluation of our results,  the 2–ΔΔCT (Livak) Method was applied. We used the weakest promoter with the lowest expression rate as calibrator for the calculations and as reference the housekeeping gene rrsD of <i>E.coli</i>.
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You can find detailed information of the qrtPCR approach [http://2012.igem.org/Team:Goettingen/Project/Methods#-.3E_Experimental_design here].<br>
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=====Results & Discussion=====
 
=====Results & Discussion=====
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[[Image:QrtPCR_ready.png|thumb|center|700px|Comparison of relative expression rates of constitutive promoters by qrtPCR and relative fluorescence (parts registry). The blue bar indicates the measured expression rates for our constructs (J23100, J23104, J23105, J23109, J23114, J23113, J23106, J23113) and the red ones those for the literature values represented in the "parts registry" for [https://parts.igem.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100]. The measurements are illustrated in a logarithmic application. The standard variation was calculated for our measured values (black error bar).]]
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[[Image:QrtPCR_ready.png|thumb|center|700px|Comparison of relative expression rates of constitutive promoters by qrtPCR and relative fluorescence (see parts registry,[https://parts.igem.org/Promoters/Catalog/Anderson Anderson family]). The blue bar indicates the measured expression rates for our constructs (J23100, J23104, J23105, J23106, J23109, J23112, J23113, J23114) and the red ones those for the literature values represented in the “parts registry”. The measurements are illustrated in a logarithmic application. The standard variation was calculated for our measured values (black error bar).]]
  
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Overall, each tested promoter construct indicated differences in expression rates in comparison to values from the “parts registry”. In fact, both data-sets were collected by methods which produce data at different points after gene expression. A common trend was detected for the strongest promoters J23100, J23104, J23105 and J23109 together with the weakest promoters J23112 and J23113. Conspicuously, the promoter J23109 revealed for qrtPCR and for relative fluorescence measurements nearly the same expression rates. The expression rates of J23114 and J23106 indicated massive differences in their expression rates and no common trend with the expression values from “parts registry”. We detected for six of our eight promoters comparable positioning in the ranking of expression rates (see Table, ranking). The promoters characterized as relative strong promoters were also in our case responsible for higher expression rates and the other way around, the promoters characterized as weak ones were in our case responsible for very low expression rates. In the case of promoter J23114 and J23106, the data-sets exhibited a completely different characterization compared to those in the “parts registry”.<br>
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As mentioned before, both datasets were collected by methods which produce data at different points after the gene expression. Quantitative real time PCR measures the amount of expressed mRNA while relative fluorescence measurements quantify on protein level. In perspective of stability and half-life periods of mRNA and proteins or due to protein modification, it is comprehensible to obtain varying data-sets and expression rates. Another problem that occurred during our quantitative real-time measurements was the deviation in some of biological replicates. This problem was also observed in another group’s experiments ([http://www.jbioleng.org/content/3/1/4 Kelly et al., 2009]). They mentioned variations across experimental conditions in the absolute activity of the BioBricks. To reduce variation in promoter activity, they measured the activity of promoters relative to BBa_J23101. Furthermore, the iGEM [https://parts.igem.org/wiki/index.php/Part:BBa_J23109 team of Groningen] which participated in 2009 also measured the relative fluorescence of TG1 strain with the promoters J23100, J23109 and J23106 via Relative Promoter Units (RPUs). Their values indicated the comparable tendency to our documented values <br>
 
For a more detailed description of our results [http://2012.igem.org/Team:Goettingen/Notebook/Results click here].
 
For a more detailed description of our results [http://2012.igem.org/Team:Goettingen/Notebook/Results click here].
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Revision as of 10:45, 26 September 2012

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Please enter how you used this part and how it worked out.

Applications of BBa_J23109

BBa_K190025 (Planning) constitutive promoter with GVP cluster

BBa_K190031 (Planning) constitutive promoter with fMT

User Reviews

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•••••

iGEM-Team Goettingen 2012

Characterization experiment by qrtPCR on BBa_J23100, BBa_J23104, BBa_J23105, BBa_J23106, BBa_J23109, BBa_J23112, BBa_J23113, BBa_J23114 by iGEM Team Göttingen (by C. Krüger and J. Kampf)

Description

We used quantitative real-time PCR as a powerful tool for quantitation of gene expression. We used this method to examine the expression rate of eight different constitutive promoter constructs from the Anderson family of the parts registry.

The reported activities of these promoters are given as the relative fluorescence of these plasmids in strain TG1 [1]. Promoter constructs were cloned into the vector pSB1C3 and expressed in E.coli BL21DE3 grown in LB-media (lysogeny broth). The measurements were performed for each construct and reference as a triplet. Additionally, we included H2O as negative control to predict possible contamination. For the evaluation of our results, the 2–ΔΔCT (Livak) Method was applied. We used the weakest promoter with the lowest expression rate as calibrator for the calculations and as reference the housekeeping gene rrsD of E.coli. You can find detailed information of the qrtPCR approach [http://2012.igem.org/Team:Goettingen/Project/Methods#-.3E_Experimental_design here].


Results & Discussion
Comparison of relative expression rates of constitutive promoters by qrtPCR and relative fluorescence (see parts registry,Anderson family). The blue bar indicates the measured expression rates for our constructs (J23100, J23104, J23105, J23106, J23109, J23112, J23113, J23114) and the red ones those for the literature values represented in the “parts registry”. The measurements are illustrated in a logarithmic application. The standard variation was calculated for our measured values (black error bar).

As mentioned before, both datasets were collected by methods which produce data at different points after the gene expression. Quantitative real time PCR measures the amount of expressed mRNA while relative fluorescence measurements quantify on protein level. In perspective of stability and half-life periods of mRNA and proteins or due to protein modification, it is comprehensible to obtain varying data-sets and expression rates. Another problem that occurred during our quantitative real-time measurements was the deviation in some of biological replicates. This problem was also observed in another group’s experiments ([http://www.jbioleng.org/content/3/1/4 Kelly et al., 2009]). They mentioned variations across experimental conditions in the absolute activity of the BioBricks. To reduce variation in promoter activity, they measured the activity of promoters relative to BBa_J23101. Furthermore, the iGEM team of Groningen which participated in 2009 also measured the relative fluorescence of TG1 strain with the promoters J23100, J23109 and J23106 via Relative Promoter Units (RPUs). Their values indicated the comparable tendency to our documented values
For a more detailed description of our results [http://2012.igem.org/Team:Goettingen/Notebook/Results click here].



•••••

iGEM Groningen 2009

We used a number of the constitutive promoter family members for testing our biobricks. The constitutive promoters show the expected level of fluorescence when transformed into E. coli TOP10 cells. Placing parts behind the promoters turned out to be relatively straight forward. We used this part in combination with several biobricks for building our constructs e.g. BBa_I750016 and BBa_K190028 were placed behind the promoters.

•••••

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

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