Difference between revisions of "Part:BBa K190015:Experience"

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===Applications of BBa_K190015===
 
===Applications of BBa_K190015===
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[https://parts.igem.org/wiki/index.php/Part:BBa_J33201 BBa_J33201]  (Available) E. coli chromosomal ars promoter with arsR repressor gene
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[https://parts.igem.org/wiki/index.php/Part:BBa_J33203 BBa_J33203] (Available) E. coli ars promoter, arsR gene and lacZ
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[https://parts.igem.org/wiki/index.php/Part:BBa_K190023 BBa_K190023]    (Planning)        E. coli ars promoter, with RBS site
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[https://parts.igem.org/wiki/index.php/Part:BBa_K190033 BBa_K190033]    (Planning)        E. coli ars promoter, GVP gene cluster
  
 
===User Reviews===
 
===User Reviews===
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Relative promoter units were calculated from RFP fluorescence upon arsenite induction of the ArsR-promoter. The promoter had an induction of 2.26 (RPU) after induction for 3hrs with 100uM NaAsO2. This is an internal concentration between 0.8-1.2uM As (as seen in Figure 1).
 
Relative promoter units were calculated from RFP fluorescence upon arsenite induction of the ArsR-promoter. The promoter had an induction of 2.26 (RPU) after induction for 3hrs with 100uM NaAsO2. This is an internal concentration between 0.8-1.2uM As (as seen in Figure 1).
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:[[Image:RFP over As conc2.png|250px]]  
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:[[Image:UptakeRPU.png|400px]]  
 
:Figure 1: RFP fluorescence increasing upon RFP expression under pArsR promoter (K190015)
 
:Figure 1: RFP fluorescence increasing upon RFP expression under pArsR promoter (K190015)
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<partinfo>BBa_K190033 AddReview 3</partinfo>
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<I>iGEM Edinburgh 2020</I>
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The part (<partinfo>BBa_K190015</partinfo>) was used in our (<partinfo>BBa_K3380603</partinfo>) construct. '''Figure 1''' shows the effect of As(III) addition 7microM on the transcription of the iSpinach fluorescent RNA aptamer in ''Cupriavidus Metallidurans'' cell-free extract.
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[[File:NewGraph.jpeg|800px|]]
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'''Figure 1.''C. Metallidurans'' cell-free extract containing the construct under the ArsR transcription factor repression expressing iSpinach''' Normalised Fluorescence over time in minutes. 100ng of DNA template was used. The T7 RNA polymerase and 7 &mu;M As(III) were added at time= 0 min. The measurements were done in triplicates. The black line exhibits fluorescence of the construct BBa_K3380500. The green line exhibits fluorescence of the BBa_K3380603 construct with 7 &mu;M As(III) and the blue line exhibits fluorescence with the BBA_K3380603 construct with no metal addition. The normalised fluorescence = (RFU of the sample- RFU blank)/ femtomole of dsDNA. The RFU blank is measured without the DNA template and the femtomole dsDNA can be calculated by knowing the transcript size and DNA concentration. The details of the construct assembly and the exact concentrations of parts used can be found under the Experience page of the similar construct BBa_K3380500
 
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<!-- DON'T DELETE --><partinfo>BBa_K190015 EndReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_K190015 EndReviews</partinfo>

Latest revision as of 22:44, 27 October 2020

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_K190015

BBa_J33201 (Available) E. coli chromosomal ars promoter with arsR repressor gene

BBa_J33203 (Available) E. coli ars promoter, arsR gene and lacZ

BBa_K190023 (Planning) E. coli ars promoter, with RBS site

BBa_K190033 (Planning) E. coli ars promoter, GVP gene cluster

User Reviews

UNIQ4a7e3abf9e9c1a54-partinfo-00000000-QINU

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iGEM Groningen 2009

The part (BBa_K190015) was used in our overall arsenic clean up E.coli to activate gas vesicle production upon arsenic detection. The promoter seemed to activate formation of vesicles without addition of arsenic in our tests. Either the ArsR regulator protein is produced in a too low amount to control unwanted expression (in witch case the gene for ArsR has to be upregulated), or the arsenic levels in our water are already sufficient for activation of expression.

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••

iGEM Groningen 2009

Relative promoter units were calculated from RFP fluorescence upon arsenite induction of the ArsR-promoter. The promoter had an induction of 2.26 (RPU) after induction for 3hrs with 100uM NaAsO2. This is an internal concentration between 0.8-1.2uM As (as seen in Figure 1).

UptakeRPU.png
Figure 1: RFP fluorescence increasing upon RFP expression under pArsR promoter (K190015)
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•••

iGEM Edinburgh 2020

The part (BBa_K190015) was used in our (BBa_K3380603) construct. Figure 1 shows the effect of As(III) addition 7microM on the transcription of the iSpinach fluorescent RNA aptamer in Cupriavidus Metallidurans cell-free extract.

NewGraph.jpeg

Figure 1.C. Metallidurans cell-free extract containing the construct under the ArsR transcription factor repression expressing iSpinach Normalised Fluorescence over time in minutes. 100ng of DNA template was used. The T7 RNA polymerase and 7 μM As(III) were added at time= 0 min. The measurements were done in triplicates. The black line exhibits fluorescence of the construct BBa_K3380500. The green line exhibits fluorescence of the BBa_K3380603 construct with 7 μM As(III) and the blue line exhibits fluorescence with the BBA_K3380603 construct with no metal addition. The normalised fluorescence = (RFU of the sample- RFU blank)/ femtomole of dsDNA. The RFU blank is measured without the DNA template and the femtomole dsDNA can be calculated by knowing the transcript size and DNA concentration. The details of the construct assembly and the exact concentrations of parts used can be found under the Experience page of the similar construct BBa_K3380500

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UNIQ4a7e3abf9e9c1a54-partinfo-00000007-QINU