Team:Warsaw/RBSmeasurement

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<div class="note">RBS Measurement</div><br />
<div class="note">RBS Measurement</div><br />
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<div><img src="https://static.igem.org/mediawiki/2011/f/f2/Tabelka.png" alt="ERROR" align="left" />
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<b>General info</b> <font color="#FF00FF">In order to fine-tune expression of genes used in our project we have conducted measurement of various ribosome binding sites included in 2010 spring distribution. Our list of measured parts includes RBSs both from Community and Anderson's collections. We have used standard measurement kit composed of promoter BBa_J23100 and GFP+terminator part BBa_I130401. All measurements were carried out using flow cytometer.</font><br /><br />
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<b>General info</b> <font color="#FF00FF">In order to fine-tune expression of genes used in our project we have conducted measurement of various ribosome binding sites included in 2011 spring distribution. Our list of measured parts includes YFP E0030 (part 723 bp  pSB1AK3), GFP E0020 (part 723bp  pSB1A2), RFP E1010 (part 681 bp  pSB2K3), mOrange E2050, MATSG—GSGTAlinkers (part 744bp  pSB2K3), GFP    E0040  GFPmut3b (720 bp pSB1A2). Also DNA of SUPERFOLDER GFP, which was included in this distribution as a bigger part with RBS and promotor. We obtained coding sequence of SUPERFOLDER GFP using PCR method.</font><br /><br />
<b>Sample preparation</b><br />
<b>Sample preparation</b><br />
To measure fluorescence of samples 3 milliliters of LB were inoculated with a colony from the plate. Cultures were vigorously shaken in 37° C overnight. The next step was to centrifuge 1,5 milliliters of liquid culture in an eppendorf tube. Pellet was then resuspended in 100 microliters of RF. Finally, prepared samples were applied on the
To measure fluorescence of samples 3 milliliters of LB were inoculated with a colony from the plate. Cultures were vigorously shaken in 37° C overnight. The next step was to centrifuge 1,5 milliliters of liquid culture in an eppendorf tube. Pellet was then resuspended in 100 microliters of RF. Finally, prepared samples were applied on the

Revision as of 11:53, 18 September 2011

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Project description


RBS Measurement

ERROR General info In order to fine-tune expression of genes used in our project we have conducted measurement of various ribosome binding sites included in 2011 spring distribution. Our list of measured parts includes YFP E0030 (part 723 bp pSB1AK3), GFP E0020 (part 723bp pSB1A2), RFP E1010 (part 681 bp pSB2K3), mOrange E2050, MATSG—GSGTAlinkers (part 744bp pSB2K3), GFP E0040 GFPmut3b (720 bp pSB1A2). Also DNA of SUPERFOLDER GFP, which was included in this distribution as a bigger part with RBS and promotor. We obtained coding sequence of SUPERFOLDER GFP using PCR method.

Sample preparation
To measure fluorescence of samples 3 milliliters of LB were inoculated with a colony from the plate. Cultures were vigorously shaken in 37° C overnight. The next step was to centrifuge 1,5 milliliters of liquid culture in an eppendorf tube. Pellet was then resuspended in 100 microliters of RF. Finally, prepared samples were applied on the fluorimeter plates. We used Corning 96 deep well plates with clear UV transparent bottom. Measurments were carried out using Tecan HS2000 pro fluorimeter with bottom read mode and using active gain optimization function. Wavlength values for individual proteins:

GFPYFPRFPmORANGESF-GFP
Excitation [nm]488514554545488
Emission [nm]509530585568508




Graphical representation of measurement

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