"
Team:Warsaw/RBSmeasurement
From 2011.igem.org
(Difference between revisions)
| Line 20: | Line 20: | ||
<div class="note">RBS Measurement</div><br /> | <div class="note">RBS Measurement</div><br /> | ||
<div><img src="https://static.igem.org/mediawiki/2011/f/f2/Tabelka.png" alt="ERROR" align="left" /> | <div><img src="https://static.igem.org/mediawiki/2011/f/f2/Tabelka.png" alt="ERROR" align="left" /> | ||
| - | <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 /> | + | <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 /> |
<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 10:25, 18 September 2011
Project description
RBS Measurement
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 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.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:
| GFP | YFP | RFP | mORANGE | SF-GFP | |
|---|---|---|---|---|---|
| Excitation [nm] | 488 | 514 | 554 | 545 | 488 |
| Emission [nm] | 509 | 530 | 585 | 568 | 508 |
Gaphical representation of measurement
