Team:Warsaw/RBSmeasurement

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<div class="note">RBS Measurement</div><br />
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<div class="note">RBS Measurement</div><br/>
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<b>General info</b><p align=justify> 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—GSGTA linkers (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.</p>
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<b>General info</b><p align=justify> We have conducted measurement of various ribosome binding sites included in 2011 spring distribution. Our list of measured parts includes YFP E0030 , GFP SUPERFOLDER , RFP E1010, mOrange E2050, GFP E0040. DNA of SUPERFOLDER GFP, was included in the distribution as a part of a bigger biobrick. We obtained coding sequence of SUPERFOLDER GFP using PCR method. All measurements were conducted in psb1A2 - a high copy number vector that was used in previous RBS measurement experiments with GFP protein.</p>
<b>Sample preparation</b><p align="justify">
<b>Sample preparation</b><p align="justify">
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To measure fluorescence of samples 3 milliliters of LB with AMP 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
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To measure fluorescence of samples 3 milliliters of LB with AMP 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 1,5 mltube. Pellet was resuspended in 100 microliters of RF. Finally, prepared samples were placed in the
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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
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fluorimeter plate. We used Corning 96 deep well plates with clear UV transparent bottom. Measurements were carried out using Tecan HS2000 pro fluorimeter with bottom read mode and using active gain optimization
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function. Wavlength values for individual proteins:</p><br />
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function. Wavelength values for individual proteins:</p><br />
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We had also found our measure depend on antibiotics resistance. We measured two of samples 3 ml of LB supplemented with chloramphenicol was inoculated with a single colony and incubated at 37oC with shaking. The charts below show the results of measuring fluorescence.
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We also conducted flow cytometry measurements for majority of our parts.
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Latest revision as of 22:42, 21 September 2011

Example Tabs


RBS Measurement

General info

We have conducted measurement of various ribosome binding sites included in 2011 spring distribution. Our list of measured parts includes YFP E0030 , GFP SUPERFOLDER , RFP E1010, mOrange E2050, GFP E0040. DNA of SUPERFOLDER GFP, was included in the distribution as a part of a bigger biobrick. We obtained coding sequence of SUPERFOLDER GFP using PCR method. All measurements were conducted in psb1A2 - a high copy number vector that was used in previous RBS measurement experiments with GFP protein.

Sample preparation

To measure fluorescence of samples 3 milliliters of LB with AMP 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 1,5 mltube. Pellet was resuspended in 100 microliters of RF. Finally, prepared samples were placed in the fluorimeter plate. We used Corning 96 deep well plates with clear UV transparent bottom. Measurements were carried out using Tecan HS2000 pro fluorimeter with bottom read mode and using active gain optimization function. Wavelength values for individual proteins:


GFPYFPRFPmORANGESF-GFP
Excitation [nm]488514554545488
Emission [nm]509530585568508
We also conducted flow cytometry measurements for majority of our parts.

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