Difference between revisions of "Part:BBa K2066045:Experience"
 
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===Characterizations of BBa_K2066045===
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First, all RBS library parts were sequenced confirmed using Sanger sequencing.
  
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In order to functionally characterize our RBS library BL21 E. coli were transformed with one of our standard characterization devices (BBa_K2066035, BBa_K2066036, BBa_K2066044-K2066049). All constructs were characterized on the pSB1C3 high-copy backbone. Cells were then selected for with antibiotics and individual colonies were grown overnight in M9 minimal media.
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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how you used this part and how it worked out.
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===Characterizations of BBa_K2066045===
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Cells were collected at log phase (OD ~ 0.4-0.6) for analysis. Culture was diluted 1:10 in 1x PBS and run on a BioRad S3e cell sorter. For each initialization of the cell sorter we also ran Spherotech RCP-30-5A Rainbow Calibration Particles to convert arbitrary fluorescence units into absolute Molecules of Equivalent Fluorescence (MEFL).
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The Alverno_Ca team characterized BBa_K2066045 as well as a series of other plasmids using TX-TL, a mean of in vitro transcription and translation using cell extract. Below is a graph of normalized expression of BBa_K2066045 in comparison to the other palsmids in the sequence.  
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https://static.igem.org/mediawiki/2016/4/4f/T--Alverno_CA--W%26M_corrected.png"
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Raw FCS files were analyzed using FlowCal from the Tabor lab to automatically gate the cell populations based on FSC and SSC (Castillo-Hair et al., 2016).  
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This graph shows the fluorescence of the plasmids, over a period of 12 and a half hours, in a plate reader.
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https://static.igem.org/mediawiki/2016/c/c1/T--Alverno_CA--W%26M_plasmid_time_traces_in_TXTL%282%29.png" alt="iGEM"
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The graph below shows the results of our characterization efforts. Solid lines are the mean of three biological replicates, and shading represents +/- standard error of the mean.  
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https://static.igem.org/mediawiki/parts/2/29/William_and_Mary_RBS_Partspage.png
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References: 
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Castillo-Hair, Sebastian M., et al. "FlowCal: A user-friendly, open source software tool for automatically converting flow cytometry data from arbitrary to calibrated units." ACS synthetic biology (2016).
  
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===Applications of BBa_K2066045===
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===Applications of BBa_K2066036===
  
 
===User Reviews===
 
===User Reviews===
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Latest revision as of 02:24, 29 October 2016

Characterizations of BBa_K2066045

First, all RBS library parts were sequenced confirmed using Sanger sequencing.

In order to functionally characterize our RBS library BL21 E. coli were transformed with one of our standard characterization devices (BBa_K2066035, BBa_K2066036, BBa_K2066044-K2066049). All constructs were characterized on the pSB1C3 high-copy backbone. Cells were then selected for with antibiotics and individual colonies were grown overnight in M9 minimal media.

Cells were collected at log phase (OD ~ 0.4-0.6) for analysis. Culture was diluted 1:10 in 1x PBS and run on a BioRad S3e cell sorter. For each initialization of the cell sorter we also ran Spherotech RCP-30-5A Rainbow Calibration Particles to convert arbitrary fluorescence units into absolute Molecules of Equivalent Fluorescence (MEFL).

Raw FCS files were analyzed using FlowCal from the Tabor lab to automatically gate the cell populations based on FSC and SSC (Castillo-Hair et al., 2016).

The graph below shows the results of our characterization efforts. Solid lines are the mean of three biological replicates, and shading represents +/- standard error of the mean.

William_and_Mary_RBS_Partspage.png


References: Castillo-Hair, Sebastian M., et al. "FlowCal: A user-friendly, open source software tool for automatically converting flow cytometry data from arbitrary to calibrated units." ACS synthetic biology (2016).

Applications of BBa_K2066036

User Reviews

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