http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=250&target=Joseph2010.igem.org - User contributions [en]2024-03-29T07:44:05ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Induction_protocols_for_GAL1_CUP1_MET17_promotersInduction protocols for GAL1 CUP1 MET17 promoters2010-10-26T10:52:08Z<p>Joseph: </p>
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<h3>Induction protocols for the GAL1, CUP1 and MET17 promoters</h3><br />
<p>Yeast cells containing the relevant transformants were inoculated into 5ml SD medium cultures. Cultures contained 2% raffinose to provide the sugar source and a combination of amino acids depending on the plasmid and selection marker (Ura, His, Met, Trp at 0.2% and Leu at 0.6%).<br><br />
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Cultures were incubated on a shaker overnight at 30oC.<br><br />
<br><br />
Samples were taken the following day and the OD600 was measured. Specific volumes of the yeast cultures were then taken and inoculated into new 5ml SD raffinose cultures that additionally contained the relevant concentration of inducer/repressor (galactose or copper sulphate or methionine).<br><br />
<br><br />
The volumes used in these inoculations were calculated so that the OD600 reading obtained prior to testing was 0.6.<br><br />
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After cultures had reached an OD600 of 0.6 samples were washed and re-suspended in PBS<br><br />
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The induction of GAL1, CUP1 and MET17 was then tested by observing the expression of associated fluorescent proteins (GFP and CFP)</p><br><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Josephhttp://2010.igem.org/Galactose_dose_response_of_Gal1_Promoter_in_pRS415Galactose dose response of Gal1 Promoter in pRS4152010-10-25T14:09:11Z<p>Joseph: Removing all content from page</p>
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<div></div>Josephhttp://2010.igem.org/Team:Aberdeen_Scotland/Galactose_dose_response_of_Gal1_Promoter_in_pRS415Team:Aberdeen Scotland/Galactose dose response of Gal1 Promoter in pRS4152010-10-25T14:08:43Z<p>Joseph: New page: {{:Team:Aberdeen_Scotland/css}} {{:Team:Aberdeen_Scotland/Title}} <h1>Measurement of dose responsiveness of the GAL1 promoter to galactose using construct GAL1p-(Npep-GFP)</h1> <h3>Aim</h3...</p>
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{{:Team:Aberdeen_Scotland/Title}}<br />
<h1>Measurement of dose responsiveness of the GAL1 promoter to galactose using construct GAL1p-(Npep-GFP)</h1><br />
<h3>Aim</h3><br />
<p><br />
Previous dose response experiments using the fluorometer revealed that full GAL1 promoter induction was achieved at concentrations above 0.5% (data not shown). We wanted to examine the dose responsive behaviour of the GAL1 promoter across a full range of concentrations. Therefore the dose response experiments were repeated using lower concentrations of this inducing agent. We have therefore tested media containing: 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, 1% and 2% of galactose. <br />
</p><br />
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<h3>Protocol</h3><br />
<p><br />
1. Yeast transformed with a plasmid carrying the GAL1p-(Npep-GFP) construct was inoculated overnight into 5 ml of synthetic defined (SD) medium with amino acids: his (0.2 %), met (0.2 %), ura (0.2 %), trp (0.2 %) and raffinose (2 %) as the carbon source. <br><br><br />
<br />
2. The following evening this cell culture was sub-cultured into a flask containing pre-warmed SD medium (50 mls) with 2% raffinose, and one of a range of concentrations of galactose between 0.05% and 2% of galactose, to achieve an optical density at 600nm of 0.6 by 9.00 am the following morning. <br><br />
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<br><br />
3. Samples were washed into PBS, and diluted 1/20 in preparation for FACS analysis.<br />
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<h3>Results</h3><br />
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Flow cytometry was used to quantify GFP fluorescence, with an excitation wavelength of 488 nm, and an emission filter of 510 nm, </p><br />
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[[Image: Gal-facs3.jpg|300 px]]<br />
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The graph above summarises the FACS data, and shows that the intensity of GFP expressing cells increases in response to the percentage of galactose in the growth medium. The GAL1 promoter in our construct showed a high degree of sensitivity to the inducing agent, with concentrations as low as 0.01% having significant inducing potential. <br />
<br><br><br />
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<h3>Conclusion</h3><br />
<p><br />
The experiment clearly showed that the percentage of cells expressing GFP was exquisitely sensitive to the presence of galactose, with the dose response saturating above 0.1% galactose. This therefore clearly shows that the GAL1 promoter is highly sensitive, but that as a synthetic biology part, it may not exhibit ideal linear responses to inducing agent for some applications. The observed GFP expression response suggests that the GAL1 promoter behaves as an analogue switch across only a very narrow range of inducer concentrations. <br />
</p><br />
<b>[[https://2010.igem.org/Team:Aberdeen_Scotland/Results Return to Results Main page]]</b><br />
<br></div>Josephhttp://2010.igem.org/Team:Aberdeen_Scotland/ResultsTeam:Aberdeen Scotland/Results2010-10-25T14:08:04Z<p>Joseph: </p>
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<h1>Main Experimental Results</h1><br />
<br />
<h2>1.Promoter characterisation</h2><br />
<br />
<h4>(a) Characterising the CUP1 promoter induction characteristics</h4><br />
<p><br />
Here, we successfully characterised the induction characteristics of the CUP1 promoter using construct CUP1-[GFP]</p><br />
<p><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Timed_Induction_of_the_CUP1_Promoter_Using_N4"><i>Timed Induction of the CUP1 Promoter Using CUP1p-GFP</i></a></p><br />
<p><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Copper_Dose_Response_of_the_CUP1_Promoter_Using_N4"><i>Copper Dose Response of the CUP1 Promoter Using CUP1p-GFP</i></a></p><br />
<br><br />
<br />
<h4> b) Characterising the GAL1 promoter induction characteristics </h4><br />
<p>Here, we successfully characterised the induction characteristics of the GAL1 promoter using construct GAL1-[GFP]</p><br />
<p><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Timed_Induction_of_Gal1_Promoter_in_pRS415"><i>Timed Induction of Gal1 Promoter using GAL1p-[Npep-GFP]</i></a></p><br />
<p><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Galactose_dose_response_of_Gal1_Promoter_in_pRS415"><i>Galactose Dose Response of Gal1 Promoter using GAL1p-[Npep-GFP]</i></a></p><br />
<br><br><br />
<br />
<br />
<h2>2.Switch characterisation</h2><br />
<br />
<h4> (a) Characterising the GAL1 promoter induction characteristics </h4><br />
<p>Here, we successfully characterised the induction characteristics of the GAL1 promoter using construct GAL1p-[Npeptide-GFP]</p><br />
<p><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Timed_Induction_of_Gal1_Promoter_in_pRS415"><i>Timed Induction of Gal1 Promoter using GAL1p-[Npep-GFP]</i></a></p><br />
<br><br />
<br />
<h4> (b) Characterising the GAL1 promoter dose-responsiveness characteristics </h4><br />
<p>Here, we successfully characterised the dose response characteristics of the GAL1 promoter using construct GAL1p-[Npeptide-GFP]</p><br />
<p><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Galactose_dose_response_of_Gal1_Promoter_in_pRS415"><i>Galactose Dose Response of Gal1 Promoter using GAL1p-[Npep-GFP]</i></a></p><br />
<br><br />
<br />
<h4> (c) Characterising the expression of MS2-CFP from the construct CUP1p-[MS2-CFP]</h4><br />
<p>Here we identified the failure of the CUP1p-[MS2-CFP] construct to direct expression of the fusion protein at significant level, using a variety of analytical techniques to show that CFP expression was undetectable under a range of conditions</p><br />
<p><a href="https://2010.igem.org/1._Confirmation_using_microscope_and_fluorometer_analysis_that_the_pRS414_construct_was_not_expressing_CFP"><i>Confirmation that CUP1p-[MS2-CFP] did not express CFP</i></a></p><br />
<br><br />
<br />
<h4> (d) Characterising the translational repression of GAL1p-[Npeptide-GFP] by trans expression of the MS2 protein.</h4><br />
<p>Here, we used <i>trans</i> expression of the MS2 protein to show that the MS2 stem loops that formed part of the 5’ leader of the GAL1p-[Npeptide-GFP] mRNA were successfully recognised by the MS2 RNA binding protein, to cause translation repression of N-pep-GFP expression, validating our RNA stem loop-based translational control approach. </p><br />
<p><a href="https://2010.igem.org/MS2_Coat-Protein_Effect_on_Expression_of_GFP_in_pRS415"><i>The effect of MS2 coat protein expresion on GAL1p-[Npep-GFP] expression</i></a></p><br />
<br><br><br />
<br />
<h2>3.Switch troubleshooting</h2><br />
<h4> (a) Cassette replacement experiment – promoter </h4> <br />
<p>Here we used homologous recombination to replace the CUP1 promoter in CUP1p-[MS2-CFP] with a previoulsy tested and functioning CUP1 promoter with 5' untranslated leader sequence <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Copper_Dose_Response_of_the_CUP1_Promoter_Using_N4"><i>CUP1 Characterisation in CUP1p-GFP</i></a> and determined that the promoter was not the faulty component in CUP1p-[MS2-CFP].</p><br />
<p><a href="https://2010.igem.org/Experimental_Layout"><i>Using homologous recombination to replace the CUP1 promoter in CUP1p-[MS2-CFP] with a CUP1 promoter plus 5' untranslated leader sequence </i></a></p><br />
<br><br />
<br />
<h4> (b) Cassette replacement experiment – fluorescent protein </h4><br />
<p>Here we replaced the GFP sequence in TEF1p -[GFP] which constitutively expresses GFP with the CFP sequence from CUP1p-[MS2-CFP] and determined that the CFP sequence was expressed properly and therefore functioning correctly. </p><br />
<p><a href="https://2010.igem.org/Experimental_Layout"><i>Using homologous recombination to replace the CFP fluorescent protein in CUP1p-[MS2-CFP] with a GFP replacement variant </i></a></p><br />
<br><br><br />
<br />
<br />
<h2>4. Other Biobrick testing </h2><br />
<h4> mOrange experiments </h4><br />
<p>In these experiments, we tested the Biobrick E2050 mOrange from the Registry of Parts and confirmed that within our gene cassette,GAL1p-[Npep-GFP]this Biobrick part did not function as expected. </p><br />
<p><a href="https://2010.igem.org/Homologous_Recombination_of_E2050_into_pRS415_Construct_in_Place_of_GFP_Protein"><i>Homologous Recombination of E2050 into GAL1p-[Npep-GFP] Construct in Place of GFP Protein </i></a></p><br />
<p><br />
<a href="https://2010.igem.org/FACS_Analysis_of_mOrange_recombinant_pRS415"><i>FACS analysis of mOrange expression under Gal1 promoter control in GAL1p-[Npep-mOrange]</i></a></p><br />
<br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Josephhttp://2010.igem.org/Timed_Induction_of_Gal1_Promoter_in_pRS415Timed Induction of Gal1 Promoter in pRS4152010-10-25T14:05:51Z<p>Joseph: Removing all content from page</p>
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<div></div>Josephhttp://2010.igem.org/Team:Aberdeen_Scotland/Timed_Induction_of_Gal1_Promoter_in_pRS415Team:Aberdeen Scotland/Timed Induction of Gal1 Promoter in pRS4152010-10-25T14:04:54Z<p>Joseph: New page: {{:Team:Aberdeen_Scotland/css}} {{:Team:Aberdeen_Scotland/Title}} <h1>Measurement of induction of the GAL1 promoter over time in construct GAL1p-(Npep-GFP)</h1> <h3>Aim</h3> <p>The aim of ...</p>
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<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<h1>Measurement of induction of the GAL1 promoter over time in construct GAL1p-(Npep-GFP)</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment was to test the response of the GAL1 promoter in the presence of galactose over time, by measuring the expression of GFP, the downstream gene. Construct Gal1p-(Npep-GFP) was used in the experiments described here <br />
</p><br />
<br />
<h3>Protocol</h3><br />
<p><br />
1. Yeast transformed with a plasmid carrying the GAL1p-(Npep-GFP) construct was inoculated overnight into 5 ml of synthetic defined (SD) medium with amino acids: his (0.2 %), met (0.2 %), ura (0.2 %), trp (0.2 %) and Raffinose (2 %) as the carbon source. <br><br><br />
<br />
2. The following evening 861 µl of this cell culture were sub-cultured into a flask containing pre-warmed SD medium (50 mls) to achieve an optical density at 600nm of 0.3 by 10am the following morning. <br><br />
<br />
<br><br />
3. At OD 600 of 0.30, a 1 ml sample was taken to represent the t=0 min sample, and then galactose addded to a final concentration of 0.1 % w/v to begin the promoter induction process. Samples were then taken every 20 minutes thereafter for a period of 170 minutes. All samples were pelleted (13000 rpm, 5min, 4 degrees C), washed once with PBS buffer and stored on ice. Once collected all samples were then dispensed in PBS and diluted by a factor of 1/20 for flow cytometry analysis.<br />
<br><br><br />
<br />
<h3>Results</h3><br />
<br />
FACS data showing the changes to the GFP expression (peak to right) and non GFP expressing cells (peak to left) over time as a result of galactose being added.<br />
<br />
There are two significant peaks. The peak to the left of the graph represents the number of cells which did not express GFP and the peak to the right the number of the cells which did express GFP. <br />
The highest peak to the left is produced by the cells before adding galactose and the peak to the right is not present thus there is no GFP expression by the cells at time zero of the experiment, hence no natural GFP expression by the cells.<br />
As time increases there is an increased number of cells which are expressing GFP in the presence of galactose inducer, shown by the gradual increase of the peak to the right over time. The visible peak starts appearing 60 mins after adding galactose (the light blue line). </p><br />
<br />
<br><br><br />
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<center><br />
[[Image: Gal-facs.jpg|300 px]]<br />
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[[Image: Gal-facs2.jpg|300 px]]<br />
</center><br />
<br><br><br />
The graph above summarises the FACS data, and shows that the intensity of GFP expressing cells increases over time after galactose has been added. The graph does not reach a plateau opver the time of the experiment. This may suggest that the cells have not expressed to their maximum capacity. Therefore to conclude, further experiments may be repeated with an increased galactose concentration or an increased period of time over which the experiment was carried out.<br />
<br><br><br />
<br />
<h3>Conclusion</h3><br />
<p><br />
The experiment clearly showed that the percentage of cells expressing GFP increased to 68% after 167 minutes from the time that 0.1 % galactose was added to the culture medium. This therefore clearly showing that galactose has successfully induced the expression of the GFP from the GAL1-(Npep-GFP) construct. Expression induction was almost linear over this time, and after 167 minutes, the expression induction had still not reached a plateau. <br />
</p><br />
<b>[[https://2010.igem.org/Team:Aberdeen_Scotland/Results Return to Results Main page]]</b><br />
<br></div>Josephhttp://2010.igem.org/Team:Aberdeen_Scotland/ResultsTeam:Aberdeen Scotland/Results2010-10-25T14:04:32Z<p>Joseph: </p>
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<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<html><br />
<h1>Main Experimental Results</h1><br />
<br />
<h2>1.Promoter characterisation</h2><br />
<br />
<h4>(a) Characterising the CUP1 promoter induction characteristics</h4><br />
<p><br />
Here, we successfully characterised the induction characteristics of the CUP1 promoter using construct CUP1-[GFP]</p><br />
<p><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Timed_Induction_of_the_CUP1_Promoter_Using_N4"><i>Timed Induction of the CUP1 Promoter Using CUP1p-GFP</i></a></p><br />
<p><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Copper_Dose_Response_of_the_CUP1_Promoter_Using_N4"><i>Copper Dose Response of the CUP1 Promoter Using CUP1p-GFP</i></a></p><br />
<br><br />
<br />
<h4> b) Characterising the GAL1 promoter induction characteristics </h4><br />
<p>Here, we successfully characterised the induction characteristics of the GAL1 promoter using construct GAL1-[GFP]</p><br />
<p><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Timed_Induction_of_Gal1_Promoter_in_pRS415"><i>Timed Induction of Gal1 Promoter using GAL1p-[Npep-GFP]</i></a></p><br />
<p><a href="https://2010.igem.org/Galactose_dose_response_of_Gal1_Promoter_in_pRS415"><i>Galactose Dose Response of Gal1 Promoter using GAL1p-[Npep-GFP]</i></a></p><br />
<br><br><br />
<br />
<br />
<h2>2.Switch characterisation</h2><br />
<br />
<h4> (a) Characterising the GAL1 promoter induction characteristics </h4><br />
<p>Here, we successfully characterised the induction characteristics of the GAL1 promoter using construct GAL1p-[Npeptide-GFP]</p><br />
<p><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Timed_Induction_of_Gal1_Promoter_in_pRS415"><i>Timed Induction of Gal1 Promoter using GAL1p-[Npep-GFP]</i></a></p><br />
<br><br />
<br />
<h4> (b) Characterising the GAL1 promoter dose-responsiveness characteristics </h4><br />
<p>Here, we successfully characterised the dose response characteristics of the GAL1 promoter using construct GAL1p-[Npeptide-GFP]</p><br />
<p><a href="https://2010.igem.org/Galactose_dose_response_of_Gal1_Promoter_in_pRS415"><i>Galactose Dose Response of Gal1 Promoter using GAL1p-[Npep-GFP]</i></a></p><br />
<br><br />
<br />
<h4> (c) Characterising the expression of MS2-CFP from the construct CUP1p-[MS2-CFP]</h4><br />
<p>Here we identified the failure of the CUP1p-[MS2-CFP] construct to direct expression of the fusion protein at significant level, using a variety of analytical techniques to show that CFP expression was undetectable under a range of conditions</p><br />
<p><a href="https://2010.igem.org/1._Confirmation_using_microscope_and_fluorometer_analysis_that_the_pRS414_construct_was_not_expressing_CFP"><i>Confirmation that CUP1p-[MS2-CFP] did not express CFP</i></a></p><br />
<br><br />
<br />
<h4> (d) Characterising the translational repression of GAL1p-[Npeptide-GFP] by trans expression of the MS2 protein.</h4><br />
<p>Here, we used <i>trans</i> expression of the MS2 protein to show that the MS2 stem loops that formed part of the 5’ leader of the GAL1p-[Npeptide-GFP] mRNA were successfully recognised by the MS2 RNA binding protein, to cause translation repression of N-pep-GFP expression, validating our RNA stem loop-based translational control approach. </p><br />
<p><a href="https://2010.igem.org/MS2_Coat-Protein_Effect_on_Expression_of_GFP_in_pRS415"><i>The effect of MS2 coat protein expresion on GAL1p-[Npep-GFP] expression</i></a></p><br />
<br><br><br />
<br />
<h2>3.Switch troubleshooting</h2><br />
<h4> (a) Cassette replacement experiment – promoter </h4> <br />
<p>Here we used homologous recombination to replace the CUP1 promoter in CUP1p-[MS2-CFP] with a previoulsy tested and functioning CUP1 promoter with 5' untranslated leader sequence <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Copper_Dose_Response_of_the_CUP1_Promoter_Using_N4"><i>CUP1 Characterisation in CUP1p-GFP</i></a> and determined that the promoter was not the faulty component in CUP1p-[MS2-CFP].</p><br />
<p><a href="https://2010.igem.org/Experimental_Layout"><i>Using homologous recombination to replace the CUP1 promoter in CUP1p-[MS2-CFP] with a CUP1 promoter plus 5' untranslated leader sequence </i></a></p><br />
<br><br />
<br />
<h4> (b) Cassette replacement experiment – fluorescent protein </h4><br />
<p>Here we replaced the GFP sequence in TEF1p -[GFP] which constitutively expresses GFP with the CFP sequence from CUP1p-[MS2-CFP] and determined that the CFP sequence was expressed properly and therefore functioning correctly. </p><br />
<p><a href="https://2010.igem.org/Experimental_Layout"><i>Using homologous recombination to replace the CFP fluorescent protein in CUP1p-[MS2-CFP] with a GFP replacement variant </i></a></p><br />
<br><br><br />
<br />
<br />
<h2>4. Other Biobrick testing </h2><br />
<h4> mOrange experiments </h4><br />
<p>In these experiments, we tested the Biobrick E2050 mOrange from the Registry of Parts and confirmed that within our gene cassette,GAL1p-[Npep-GFP]this Biobrick part did not function as expected. </p><br />
<p><a href="https://2010.igem.org/Homologous_Recombination_of_E2050_into_pRS415_Construct_in_Place_of_GFP_Protein"><i>Homologous Recombination of E2050 into GAL1p-[Npep-GFP] Construct in Place of GFP Protein </i></a></p><br />
<p><br />
<a href="https://2010.igem.org/FACS_Analysis_of_mOrange_recombinant_pRS415"><i>FACS analysis of mOrange expression under Gal1 promoter control in GAL1p-[Npep-mOrange]</i></a></p><br />
<br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Josephhttp://2010.igem.org/Copper_Dose_Response_of_the_CUP1_Promoter_Using_N4Copper Dose Response of the CUP1 Promoter Using N42010-10-25T13:59:51Z<p>Joseph: Removing all content from page</p>
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<div></div>Josephhttp://2010.igem.org/Team:Aberdeen_Scotland/Copper_Dose_Response_of_the_CUP1_Promoter_Using_N4Team:Aberdeen Scotland/Copper Dose Response of the CUP1 Promoter Using N42010-10-25T13:59:01Z<p>Joseph: New page: {{:Team:Aberdeen_Scotland/css}} {{:Team:Aberdeen_Scotland/Title}} <html> <h1>Quantitative Determination of the Response of the CUP1p - [GFP] to Varying Concentration of CuSO4</h1> <h3>Aim...</p>
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<h1>Quantitative Determination of the Response of the CUP1p - [GFP] to Varying Concentration of CuSO4</h1><br />
<br />
<h3>Aim</h3><br />
<p>The aim of this experiment is to characterise the CUP1 promoter present in CUP1p - [GFP] by determining whether<br />
it displays a dose response quality. The determined characteristics of this promoter can then be applied to the promoter present in CUP1p - [MS2-CFP] in order to allow more precise modelling of the switch.</p><br />
<br />
<h3>Hypothesis</h3><br />
<p>The CUP1 promoter exhibits a linear relationship between the concentration of CuSO4 and the level of GFP expression when CuSO4 concentrations range from 0µM to 100µM. At concentrations of 100µM and higher the expression level of GFP will have reached a steady state and will remain unchanged despite increasing CuSO4 concentrations.</p><br />
<br />
<h3>Protocol</h3><br />
<p>A genomically integrated GFP gene under control of a CUP1 promoter was used to characterise the control properties of this promoter. This construct is referred to as CUP1p-[GFP] and was transformed into the yeast strain BY4741 for analysis.<br />
<br><br />
Three separate starter cultures were prepared of CUP1p-[GFP] in 5mL SD medium. The following tubes were then prepared in triplicate.</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/3/3a/Table1_for_Quantitative_determination_of_the_response_of_the_CUP1..._.png"/><br />
</center><br />
<p>Each one of these tubes was inoculated with N4 from each stater culture in order to provide triplicates of each concentratuion value. The cells were later harvested once the OD600 had reached 0.6. Triplicates from each culture were then loaded onto a 96 microtitre plate. The fluorescence of each sample was measured using a fluorometer running the "RussGFP" protocol.</p><br />
<h3>Results</h3><br />
<p>For full data spreadsheet<br><br />
IGEM 240610 JH+SL PCup Induction.xslx</p><br><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/2/20/Table2_for_Quantitative_determination_of_..._.png"/><br />
</center><br />
</p><br />
<h3>Conclusion</h3><br />
<p>The data suggests evidence of a dose dependent response. We can see from Figure 1 that the relationship between the GFP levels and the copper concentrations is linear when the concentrations of CuSO4 range from 0µM to 75µM. At concentrations higher than 75µM the response seems to reach a plateau and the GFP levels no longer increase along with increasing copper concentrations. Figure 2 shows us that these characteristics appear for each individual culture.</p><br />
<p>The data supports the hypothesis that the response of the copper promoter is dose dependent for a defined range of copper concentrations. The data however indicates that this range is smaller than our initial hypothesis suggested and that a plateau is reached with concentrations of 75μM as opposed to the initial 100μM.</p><br />
<h3>References</h3><br />
<p>[1]. Gorman JA, Clark PE, Lee MC, Debouck C and Rosenberg M<br><br />
Regulation of the yeast metallothionein gene<br><br />
Gene, 48 (1986 13-22)<br><br />
<br><br />
</html><br />
<br />
<b>[[https://2010.igem.org/Team:Aberdeen_Scotland/Results Return to Results Main page]]</b></div>Josephhttp://2010.igem.org/Team:Aberdeen_Scotland/ResultsTeam:Aberdeen Scotland/Results2010-10-25T13:58:23Z<p>Joseph: </p>
<hr />
<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<html><br />
<h1>Main Experimental Results</h1><br />
<br />
<h2>1.Promoter characterisation</h2><br />
<br />
<h4>(a) Characterising the CUP1 promoter induction characteristics</h4><br />
<p><br />
Here, we successfully characterised the induction characteristics of the CUP1 promoter using construct CUP1-[GFP]</p><br />
<p><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Timed_Induction_of_the_CUP1_Promoter_Using_N4"><i>Timed Induction of the CUP1 Promoter Using CUP1p-GFP</i></a></p><br />
<p><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Copper_Dose_Response_of_the_CUP1_Promoter_Using_N4"><i>Copper Dose Response of the CUP1 Promoter Using CUP1p-GFP</i></a></p><br />
<br><br />
<br />
<h4> b) Characterising the GAL1 promoter induction characteristics </h4><br />
<p>Here, we successfully characterised the induction characteristics of the GAL1 promoter using construct GAL1-[GFP]</p><br />
<p><a href="https://2010.igem.org/Timed_Induction_of_Gal1_Promoter_in_pRS415"><i>Timed Induction of Gal1 Promoter using GAL1p-[Npep-GFP]</i></a></p><br />
<p><a href="https://2010.igem.org/Galactose_dose_response_of_Gal1_Promoter_in_pRS415"><i>Galactose Dose Response of Gal1 Promoter using GAL1p-[Npep-GFP]</i></a></p><br />
<br><br><br />
<br />
<br />
<h2>2.Switch characterisation</h2><br />
<br />
<h4> (a) Characterising the GAL1 promoter induction characteristics </h4><br />
<p>Here, we successfully characterised the induction characteristics of the GAL1 promoter using construct GAL1p-[Npeptide-GFP]</p><br />
<p><a href="https://2010.igem.org/Timed_Induction_of_Gal1_Promoter_in_pRS415"><i>Timed Induction of Gal1 Promoter using GAL1p-[Npep-GFP]</i></a></p><br />
<br><br />
<br />
<h4> (b) Characterising the GAL1 promoter dose-responsiveness characteristics </h4><br />
<p>Here, we successfully characterised the dose response characteristics of the GAL1 promoter using construct GAL1p-[Npeptide-GFP]</p><br />
<p><a href="https://2010.igem.org/Galactose_dose_response_of_Gal1_Promoter_in_pRS415"><i>Galactose Dose Response of Gal1 Promoter using GAL1p-[Npep-GFP]</i></a></p><br />
<br><br />
<br />
<h4> (c) Characterising the expression of MS2-CFP from the construct CUP1p-[MS2-CFP]</h4><br />
<p>Here we identified the failure of the CUP1p-[MS2-CFP] construct to direct expression of the fusion protein at significant level, using a variety of analytical techniques to show that CFP expression was undetectable under a range of conditions</p><br />
<p><a href="https://2010.igem.org/1._Confirmation_using_microscope_and_fluorometer_analysis_that_the_pRS414_construct_was_not_expressing_CFP"><i>Confirmation that CUP1p-[MS2-CFP] did not express CFP</i></a></p><br />
<br><br />
<br />
<h4> (d) Characterising the translational repression of GAL1p-[Npeptide-GFP] by trans expression of the MS2 protein.</h4><br />
<p>Here, we used <i>trans</i> expression of the MS2 protein to show that the MS2 stem loops that formed part of the 5’ leader of the GAL1p-[Npeptide-GFP] mRNA were successfully recognised by the MS2 RNA binding protein, to cause translation repression of N-pep-GFP expression, validating our RNA stem loop-based translational control approach. </p><br />
<p><a href="https://2010.igem.org/MS2_Coat-Protein_Effect_on_Expression_of_GFP_in_pRS415"><i>The effect of MS2 coat protein expresion on GAL1p-[Npep-GFP] expression</i></a></p><br />
<br><br><br />
<br />
<h2>3.Switch troubleshooting</h2><br />
<h4> (a) Cassette replacement experiment – promoter </h4> <br />
<p>Here we used homologous recombination to replace the CUP1 promoter in CUP1p-[MS2-CFP] with a previoulsy tested and functioning CUP1 promoter with 5' untranslated leader sequence <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Copper_Dose_Response_of_the_CUP1_Promoter_Using_N4"><i>CUP1 Characterisation in CUP1p-GFP</i></a> and determined that the promoter was not the faulty component in CUP1p-[MS2-CFP].</p><br />
<p><a href="https://2010.igem.org/Experimental_Layout"><i>Using homologous recombination to replace the CUP1 promoter in CUP1p-[MS2-CFP] with a CUP1 promoter plus 5' untranslated leader sequence </i></a></p><br />
<br><br />
<br />
<h4> (b) Cassette replacement experiment – fluorescent protein </h4><br />
<p>Here we replaced the GFP sequence in TEF1p -[GFP] which constitutively expresses GFP with the CFP sequence from CUP1p-[MS2-CFP] and determined that the CFP sequence was expressed properly and therefore functioning correctly. </p><br />
<p><a href="https://2010.igem.org/Experimental_Layout"><i>Using homologous recombination to replace the CFP fluorescent protein in CUP1p-[MS2-CFP] with a GFP replacement variant </i></a></p><br />
<br><br><br />
<br />
<br />
<h2>4. Other Biobrick testing </h2><br />
<h4> mOrange experiments </h4><br />
<p>In these experiments, we tested the Biobrick E2050 mOrange from the Registry of Parts and confirmed that within our gene cassette,GAL1p-[Npep-GFP]this Biobrick part did not function as expected. </p><br />
<p><a href="https://2010.igem.org/Homologous_Recombination_of_E2050_into_pRS415_Construct_in_Place_of_GFP_Protein"><i>Homologous Recombination of E2050 into GAL1p-[Npep-GFP] Construct in Place of GFP Protein </i></a></p><br />
<p><br />
<a href="https://2010.igem.org/FACS_Analysis_of_mOrange_recombinant_pRS415"><i>FACS analysis of mOrange expression under Gal1 promoter control in GAL1p-[Npep-mOrange]</i></a></p><br />
<br />
<br><br><br />
<hr><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Josephhttp://2010.igem.org/Timed_Induction_of_the_CUP1_Promoter_Using_N4Timed Induction of the CUP1 Promoter Using N42010-10-25T13:50:13Z<p>Joseph: Removing all content from page</p>
<hr />
<div></div>Josephhttp://2010.igem.org/Team:Aberdeen_Scotland/Timed_Induction_of_the_CUP1_Promoter_Using_N4Team:Aberdeen Scotland/Timed Induction of the CUP1 Promoter Using N42010-10-25T13:49:39Z<p>Joseph: New page: {{:Team:Aberdeen_Scotland/css}} {{:Team:Aberdeen_Scotland/Title}} <html> <h1>Timed Induction of GFP Expression by the CUP1p-[GFP]</h1> <h3>Aim</h3> <p>The aim of this experiment is to det...</p>
<hr />
<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<html><br />
<br />
<h1>Timed Induction of GFP Expression by the CUP1p-[GFP]</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment is to determine how quickly the CUP1 promoter is fully induced by set concentrations of copper by measuring the levels of GFP being expressed by the yeast cells.This is done using the CUP1p-[GFP] construct.</p><br />
<h3>Hypothesis</h3><br />
<p>The CUP1 promoter will rapidly become fully induced following exposure to set concentrations of copper. Once it is fully induced the level of GFP expression will stabilise and will no longer increase despite more time passing.</p><br />
<h3>Protocol</h3><br />
<p>A genomically integrated GFP gene under control of a CUP1 promoter in the construct CUP1p-[GFP], was used to characterise the control properties of this promoter. This construct was transformed into the yeast strain BY4741 for analysis.<br><br><br />
Starter cultures of CUP1p-[GFP] in 5mL of SD medium + Raffinose were set up and incubated overnight. A 50mL flask containing SD Raff was then inoculated using the starter cultures and incubated overnight until the OD600 reached 0.3.<br><br><br />
At t=0 a sample from the flask was put on ice to provide a background reading of yeast’s natural fluorescence without any inducer. Copper was then added to the flask at a concentration of 100μM. Samples were then taken every 20 minutes and put on ice. All samples were then normalised to an OD600 of 0.75 and were washed and re-suspended in PBS before being analysed in the fluorometer (the programme RussGFP” was used for the analysis).</p><br />
<h3>Results</h3><br />
For full data spreadsheet<br><br />
iGEM 2.6.10 JH+SL N4 Timed Induction.xslx<br><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/a/ae/PCUP1_response_graph.jpeg"/><br />
</center><br />
</p><br />
<h3>Conclusion</h3><br />
<p>Figure 1 indicates that full induction was reached after approximately 80 minutes and that the level of GFP expression seems to reach a plateau after this and no longer increases. This supports our initial hypothesis.</p><br />
</html><br />
<br />
<b>[[https://2010.igem.org/Team:Aberdeen_Scotland/Results Return to Results Main page]]</b></div>Josephhttp://2010.igem.org/Team:Aberdeen_Scotland/ResultsTeam:Aberdeen Scotland/Results2010-10-25T13:49:05Z<p>Joseph: </p>
<hr />
<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<html><br />
<h1>Main Experimental Results</h1><br />
<br />
<h2>1.Promoter characterisation</h2><br />
<br />
<h4>(a) Characterising the CUP1 promoter induction characteristics</h4><br />
<p><br />
Here, we successfully characterised the induction characteristics of the CUP1 promoter using construct CUP1-[GFP]</p><br />
<p><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Timed_Induction_of_the_CUP1_Promoter_Using_N4"><i>Timed Induction of the CUP1 Promoter Using CUP1p-GFP</i></a></p><br />
<p><a href="https://2010.igem.org/Copper_Dose_Response_of_the_CUP1_Promoter_Using_N4"><i>Copper Dose Response of the CUP1 Promoter Using CUP1p-GFP</i></a></p><br />
<br><br />
<br />
<h4> b) Characterising the GAL1 promoter induction characteristics </h4><br />
<p>Here, we successfully characterised the induction characteristics of the GAL1 promoter using construct GAL1-[GFP]</p><br />
<p><a href="https://2010.igem.org/Timed_Induction_of_Gal1_Promoter_in_pRS415"><i>Timed Induction of Gal1 Promoter using GAL1p-[Npep-GFP]</i></a></p><br />
<p><a href="https://2010.igem.org/Galactose_dose_response_of_Gal1_Promoter_in_pRS415"><i>Galactose Dose Response of Gal1 Promoter using GAL1p-[Npep-GFP]</i></a></p><br />
<br><br><br />
<br />
<br />
<h2>2.Switch characterisation</h2><br />
<br />
<h4> (a) Characterising the GAL1 promoter induction characteristics </h4><br />
<p>Here, we successfully characterised the induction characteristics of the GAL1 promoter using construct GAL1p-[Npeptide-GFP]</p><br />
<p><a href="https://2010.igem.org/Timed_Induction_of_Gal1_Promoter_in_pRS415"><i>Timed Induction of Gal1 Promoter using GAL1p-[Npep-GFP]</i></a></p><br />
<br><br />
<br />
<h4> (b) Characterising the GAL1 promoter dose-responsiveness characteristics </h4><br />
<p>Here, we successfully characterised the dose response characteristics of the GAL1 promoter using construct GAL1p-[Npeptide-GFP]</p><br />
<p><a href="https://2010.igem.org/Galactose_dose_response_of_Gal1_Promoter_in_pRS415"><i>Galactose Dose Response of Gal1 Promoter using GAL1p-[Npep-GFP]</i></a></p><br />
<br><br />
<br />
<h4> (c) Characterising the expression of MS2-CFP from the construct CUP1p-[MS2-CFP]</h4><br />
<p>Here we identified the failure of the CUP1p-[MS2-CFP] construct to direct expression of the fusion protein at significant level, using a variety of analytical techniques to show that CFP expression was undetectable under a range of conditions</p><br />
<p><a href="https://2010.igem.org/1._Confirmation_using_microscope_and_fluorometer_analysis_that_the_pRS414_construct_was_not_expressing_CFP"><i>Confirmation that CUP1p-[MS2-CFP] did not express CFP</i></a></p><br />
<br><br />
<br />
<h4> (d) Characterising the translational repression of GAL1p-[Npeptide-GFP] by trans expression of the MS2 protein.</h4><br />
<p>Here, we used <i>trans</i> expression of the MS2 protein to show that the MS2 stem loops that formed part of the 5’ leader of the GAL1p-[Npeptide-GFP] mRNA were successfully recognised by the MS2 RNA binding protein, to cause translation repression of N-pep-GFP expression, validating our RNA stem loop-based translational control approach. </p><br />
<p><a href="https://2010.igem.org/MS2_Coat-Protein_Effect_on_Expression_of_GFP_in_pRS415"><i>The effect of MS2 coat protein expresion on GAL1p-[Npep-GFP] expression</i></a></p><br />
<br><br><br />
<br />
<h2>3.Switch troubleshooting</h2><br />
<h4> (a) Cassette replacement experiment – promoter </h4> <br />
<p>Here we used homologous recombination to replace the CUP1 promoter in CUP1p-[MS2-CFP] with a previoulsy tested and functioning CUP1 promoter with 5' untranslated leader sequence <a href="https://2010.igem.org/Copper_Dose_Response_of_the_CUP1_Promoter_Using_N4"><i>CUP1 Characterisation in CUP1p-GFP</i></a> and determined that the promoter was not the faulty component in CUP1p-[MS2-CFP].</p><br />
<p><a href="https://2010.igem.org/Experimental_Layout"><i>Using homologous recombination to replace the CUP1 promoter in CUP1p-[MS2-CFP] with a CUP1 promoter plus 5' untranslated leader sequence </i></a></p><br />
<br><br />
<br />
<h4> (b) Cassette replacement experiment – fluorescent protein </h4><br />
<p>Here we replaced the GFP sequence in TEF1p -[GFP] which constitutively expresses GFP with the CFP sequence from CUP1p-[MS2-CFP] and determined that the CFP sequence was expressed properly and therefore functioning correctly. </p><br />
<p><a href="https://2010.igem.org/Experimental_Layout"><i>Using homologous recombination to replace the CFP fluorescent protein in CUP1p-[MS2-CFP] with a GFP replacement variant </i></a></p><br />
<br><br><br />
<br />
<br />
<h2>4. Other Biobrick testing </h2><br />
<h4> mOrange experiments </h4><br />
<p>In these experiments, we tested the Biobrick E2050 mOrange from the Registry of Parts and confirmed that within our gene cassette,GAL1p-[Npep-GFP]this Biobrick part did not function as expected. </p><br />
<p><a href="https://2010.igem.org/Homologous_Recombination_of_E2050_into_pRS415_Construct_in_Place_of_GFP_Protein"><i>Homologous Recombination of E2050 into GAL1p-[Npep-GFP] Construct in Place of GFP Protein </i></a></p><br />
<p><br />
<a href="https://2010.igem.org/FACS_Analysis_of_mOrange_recombinant_pRS415"><i>FACS analysis of mOrange expression under Gal1 promoter control in GAL1p-[Npep-mOrange]</i></a></p><br />
<br />
<br><br><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Josephhttp://2010.igem.org/Galactose_dose_response_of_Gal1_Promoter_in_pRS415Galactose dose response of Gal1 Promoter in pRS4152010-10-25T13:35:40Z<p>Joseph: </p>
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<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<h1>Measurement of dose responsiveness of the GAL1 promoter to galactose using construct GAL1p-(Npep-GFP)</h1><br />
<h3>Aim</h3><br />
<p><br />
Previous dose response experiments using the fluorometer revealed that full GAL1 promoter induction was achieved at concentrations above 0.5% (data not shown). We wanted to examine the dose responsive behaviour of the GAL1 promoter across a full range of concentrations. Therefore the dose response experiments were repeated using lower concentrations of this inducing agent. We have therefore tested media containing: 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, 1% and 2% of galactose. <br />
</p><br />
<br />
<h3>Protocol</h3><br />
<p><br />
1. Yeast transformed with a plasmid carrying the GAL1p-(Npep-GFP) construct was inoculated overnight into 5 ml of synthetic defined (SD) medium with amino acids: his (0.2 %), met (0.2 %), ura (0.2 %), trp (0.2 %) and raffinose (2 %) as the carbon source. <br><br><br />
<br />
2. The following evening this cell culture was sub-cultured into a flask containing pre-warmed SD medium (50 mls) with 2% raffinose, and one of a range of concentrations of galactose between 0.05% and 2% of galactose, to achieve an optical density at 600nm of 0.6 by 9.00 am the following morning. <br><br />
<br />
<br><br />
3. Samples were washed into PBS, and diluted 1/20 in preparation for FACS analysis.<br />
<br><br><br />
<br />
<h3>Results</h3><br />
<br />
Flow cytometry was used to quantify GFP fluorescence, with an excitation wavelength of 488 nm, and an emission filter of 510 nm, </p><br />
<br />
<br><br><br />
<center><br />
[[Image: Gal-facs3.jpg|300 px]]<br />
</center><br />
<br><br><br />
<br />
The graph above summarises the FACS data, and shows that the intensity of GFP expressing cells increases in response to the percentage of galactose in the growth medium. The GAL1 promoter in our construct showed a high degree of sensitivity to the inducing agent, with concentrations as low as 0.01% having significant inducing potential. <br />
<br><br><br />
<br />
<h3>Conclusion</h3><br />
<p><br />
The experiment clearly showed that the percentage of cells expressing GFP was exquisitely sensitive to the presence of galactose, with the dose response saturating above 0.1% galactose. This therefore clearly shows that the GAL1 promoter is highly sensitive, but that as a synthetic biology part, it may not exhibit ideal linear responses to inducing agent for some applications. The observed GFP expression response suggests that the GAL1 promoter behaves as an analogue switch across only a very narrow range of inducer concentrations. <br />
</p><br />
<b>[[https://2010.igem.org/Team:Aberdeen_Scotland/Results Return to Results Main page]]</b><br />
<br></div>Josephhttp://2010.igem.org/Timed_Induction_of_Gal1_Promoter_in_pRS415Timed Induction of Gal1 Promoter in pRS4152010-10-25T13:34:45Z<p>Joseph: </p>
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<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<h1>Measurement of induction of the GAL1 promoter over time in construct GAL1p-(Npep-GFP)</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment was to test the response of the GAL1 promoter in the presence of galactose over time, by measuring the expression of GFP, the downstream gene. Construct Gal1p-(Npep-GFP) was used in the experiments described here <br />
</p><br />
<br />
<h3>Protocol</h3><br />
<p><br />
1. Yeast transformed with a plasmid carrying the GAL1p-(Npep-GFP) construct was inoculated overnight into 5 ml of synthetic defined (SD) medium with amino acids: his (0.2 %), met (0.2 %), ura (0.2 %), trp (0.2 %) and Raffinose (2 %) as the carbon source. <br><br><br />
<br />
2. The following evening 861 µl of this cell culture were sub-cultured into a flask containing pre-warmed SD medium (50 mls) to achieve an optical density at 600nm of 0.3 by 10am the following morning. <br><br />
<br />
<br><br />
3. At OD 600 of 0.30, a 1 ml sample was taken to represent the t=0 min sample, and then galactose addded to a final concentration of 0.1 % w/v to begin the promoter induction process. Samples were then taken every 20 minutes thereafter for a period of 170 minutes. All samples were pelleted (13000 rpm, 5min, 4 degrees C), washed once with PBS buffer and stored on ice. Once collected all samples were then dispensed in PBS and diluted by a factor of 1/20 for flow cytometry analysis.<br />
<br><br><br />
<br />
<h3>Results</h3><br />
<br />
FACS data showing the changes to the GFP expression (peak to right) and non GFP expressing cells (peak to left) over time as a result of galactose being added.<br />
<br />
There are two significant peaks. The peak to the left of the graph represents the number of cells which did not express GFP and the peak to the right the number of the cells which did express GFP. <br />
The highest peak to the left is produced by the cells before adding galactose and the peak to the right is not present thus there is no GFP expression by the cells at time zero of the experiment, hence no natural GFP expression by the cells.<br />
As time increases there is an increased number of cells which are expressing GFP in the presence of galactose inducer, shown by the gradual increase of the peak to the right over time. The visible peak starts appearing 60 mins after adding galactose (the light blue line). </p><br />
<br />
<br><br><br />
<br />
<center><br />
[[Image: Gal-facs.jpg|300 px]]<br />
<br />
<br><br><br />
[[Image: Gal-facs2.jpg|300 px]]<br />
</center><br />
<br><br><br />
The graph above summarises the FACS data, and shows that the intensity of GFP expressing cells increases over time after galactose has been added. The graph does not reach a plateau opver the time of the experiment. This may suggest that the cells have not expressed to their maximum capacity. Therefore to conclude, further experiments may be repeated with an increased galactose concentration or an increased period of time over which the experiment was carried out.<br />
<br><br><br />
<br />
<h3>Conclusion</h3><br />
<p><br />
The experiment clearly showed that the percentage of cells expressing GFP increased to 68% after 167 minutes from the time that 0.1 % galactose was added to the culture medium. This therefore clearly showing that galactose has successfully induced the expression of the GFP from the GAL1-(Npep-GFP) construct. Expression induction was almost linear over this time, and after 167 minutes, the expression induction had still not reached a plateau. <br />
</p><br />
<b>[[https://2010.igem.org/Team:Aberdeen_Scotland/Results Return to Results Main page]]</b><br />
<br></div>Josephhttp://2010.igem.org/MS2_Coat-Protein_Effect_on_Expression_of_GFP_in_pRS415MS2 Coat-Protein Effect on Expression of GFP in pRS4152010-10-24T13:07:14Z<p>Joseph: </p>
<hr />
<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<h1> Characterising the translational repression of GAL1p-[Npeptide-GFP] by trans expression of the MS2 protein </h1><br />
<h3>Aim</h3><br />
<p>The characterisation of the effect of MS2 on the expression of GFP by GAL1p-[Npeptide-GFP] will allow more accurate modelling of the system and will allow us to determine with more precision the probability of success of the cross-inhibition of the switch. Expressing MS2 using MET17p - [MS2] will allow us to monitor the effect of MS2 without the complication of the λ-N-peptide produced by GAL1p-[Npeptide-GFP] in turn inhibiting the expression of MS2.</p><br />
<h3>Hypothesis</h3> <br />
<p>The expression of MS2 by MET17p - [MS2]will result in a decrease in the level of expression of GFP by GAL1p-[Npeptide-GFP]. The inhibition will show a linear correlation with the level of expression of MS2.</p><br />
<h3>Protocol</h3><br />
<p>During this experiment double transformants of BY4742 containing GAL1p-[Npeptide-GFP] and MET17p - [MS2] were used. Single transformants of BY4742, containing only GAL1p-[Npeptide-GFP], were used to provide the negative and positive controls for the expression of GFP.</p><br><br />
<p>The double transformants were first cultured overnight in specific conditions in order to establish the desired pre-conditions. The cells were then washed and re-cultured in a different specific set of conditions which would allow the characterisation of the effect of MS2.</p><br><br />
<center><br />
https://static.igem.org/mediawiki/2010/0/04/Conditions_for_FACS_analysis_of_MS2vsGFP.jpg<br />
</center><br />
<p>* 500μM Met was used as this concentration was been used in other experiments to successfully completely switch of the Met17 promoter [1]. <br><br />
<br><br />
The different pre-established conditions allow us to determine whether the history of the sample affects the final result.<br><br />
Final samples were then washed and normalised before being analysed using microscopy, Fluospar Optima readings and FACS analysis.</p><br><br />
<h3>Results</h3><br />
<br />
'''<u>Microscopy</u>'''<br><br />
<p>The microscopy analysis revealed that, in none of the samples, the GFP expression had been completely inhibited. All samples (bar the negative control) showed green fluorescence. The microscope did not allow us to determine if there was any variation however in the levels of GFP in each specific sample. </p><br />
<center><br />
https://static.igem.org/mediawiki/2010/c/c0/MS2vsGFP_fluorescent_cells.jpg<br />
</center><br />
'''<u>Fluospar Optima Readings</u>'''<br><br><br />
The fluorimeter readings correlated the microscopy results by recording fluorescence in all samples except the – control.<br><br />
<center><br />
https://static.igem.org/mediawiki/2010/5/5a/MS2vsGFP_fluorimeter.jpg<br />
</center><br />
<br><br />
<p>The recorded fluorescence values for the respective samples showed that there was indeed some variation in the levels of GFP (Fig 1). In both the ‘MS2 Dom’ and the ‘Race’ sample the GFP level was lower than in the + control indicating that the expression of GFP had indeed been inhibited (a 20% decrease for the ‘Race’ sample and an 11% decrease for the ‘MS2’ sample). The ‘GFP Dom’ sample however showed an approximate 8% increase in GFP fluorescence when compared to the + control. Although this is a bit unexpected is could be due to the fact that the GFP expression was initiated in the 1˚ set of conditions whereas it took place in the 2˚ for the + control. However it appears that no inhibition took place indicating that once GFP is being expressed the amount present of MS2 as expressed by MET17p - [MS2] is not able to significantly inhibit the level of GFP fluorescence.</p><br />
<br><br />
'''<u>FACS analysis</u>'''<br><br />
<p>The first FACS analysis experiment involved running samples from the same initial cultures (see Table 1). The results showed that the presence of the MS2 coat protein was having an effect on the expression of GFP. All three test samples revealed lower levels of GFP when compared to the positive control indicating that MS2 was inhibiting the expression of GFP (see Fig 3). As expected the sample where GFP had been allowed to dominate prior to expression of MS2 (GFPdom sample) showed the highest level of GFP in the test samples and equally the sample where MS2 dominated prior to the expression of GFP (MS2do sample) showed the lowest level of GFP expression.</p><br><br />
<center><br />
https://static.igem.org/mediawiki/2010/a/a5/MS2vsGFP_FACS_1.jpg<br />
</center><br><br />
<p>The second FACS analysis experiment was aimed to determine whether the inhibition of GFP expression was in any way dependent on the levels of MS2. The following cultures were set up containing transformants containing both GAL1p-[Npeptide-GFP] and MET17p - [MS2] with varying amounts of Methionine. The reasoning is that the varying levels of methionine will translate into varying amounts of MS2 being produced as the Met17 is repressed.</p><br><br />
<center><br />
https://static.igem.org/mediawiki/2010/d/d8/MS2vsGFP_FACS_2.jpg<br />
</center><br><br />
<br><br />
<p>The results showed that the inhibition of GFP expression by GAL1p-[Npeptide-GFP] by MS2 previously seen (see Fig.3) is indeed dependant on the concentration of MS2.<br><br />
<br><br />
We can see a linear relationship between GFP levels and MS2 concentrations (see Fig.4). The observed level of GFP is at its lowest with no methionine being present. No methionine present translates as the Met17 promoter being unrepressed meaning that the MS2 expression rate is at its maximum. The levels of GFP gradually increase along with an increasing concentration of methionine (this translates as the Met17 promoter gradually being repressed until MS2 is no longer being expressed).</p><br />
<br />
<h3>References</h3><br />
<br />
<p>[1] Dominik Mumberg, Rolf MulIer and Martin Funk*<br><br />
Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression<br><br />
Nucleic Acids Research, 1994, Vol. 22, No. 25 5767-5768</p><br><br />
<br><br />
<b>[[https://2010.igem.org/Team:Aberdeen_Scotland/Results Return to Results Main page]]</b></div>Josephhttp://2010.igem.org/File:MS2vsGFP_FACS_2.jpgFile:MS2vsGFP FACS 2.jpg2010-10-24T13:01:06Z<p>Joseph: </p>
<hr />
<div></div>Josephhttp://2010.igem.org/File:MS2vsGFP_FACS_1.jpgFile:MS2vsGFP FACS 1.jpg2010-10-24T12:58:55Z<p>Joseph: </p>
<hr />
<div></div>Josephhttp://2010.igem.org/MS2_Coat-Protein_Effect_on_Expression_of_GFP_in_pRS415MS2 Coat-Protein Effect on Expression of GFP in pRS4152010-10-24T12:57:43Z<p>Joseph: </p>
<hr />
<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<h1> Characterising the translational repression of GAL1p-[Npeptide-GFP] by trans expression of the MS2 protein </h1><br />
<h3>Aim</h3><br />
<p>The characterisation of the effect of MS2 on the expression of GFP by GAL1p-[Npeptide-GFP] will allow more accurate modelling of the system and will allow us to determine with more precision the probability of success of the cross-inhibition of the switch. Expressing MS2 using MET17p - [MS2] will allow us to monitor the effect of MS2 without the complication of the λ-N-peptide produced by GAL1p-[Npeptide-GFP] in turn inhibiting the expression of MS2.</p><br />
<h3>Hypothesis</h3> <br />
<p>The expression of MS2 by MET17p - [MS2]will result in a decrease in the level of expression of GFP by GAL1p-[Npeptide-GFP]. The inhibition will show a linear correlation with the level of expression of MS2.</p><br />
<h3>Protocol</h3><br />
<p>During this experiment double transformants of BY4742 containing GAL1p-[Npeptide-GFP] and MET17p - [MS2] were used. Single transformants of BY4742, containing only GAL1p-[Npeptide-GFP], were used to provide the negative and positive controls for the expression of GFP.</p><br><br />
<p>The double transformants were first cultured overnight in specific conditions in order to establish the desired pre-conditions. The cells were then washed and re-cultured in a different specific set of conditions which would allow the characterisation of the effect of MS2.</p><br><br />
<center><br />
https://static.igem.org/mediawiki/2010/0/04/Conditions_for_FACS_analysis_of_MS2vsGFP.jpg<br />
</center><br />
<p>* 500μM Met was used as this concentration was been used in other experiments to successfully completely switch of the Met17 promoter [1]. <br><br />
<br><br />
The different pre-established conditions allow us to determine whether the history of the sample affects the final result.<br><br />
Final samples were then washed and normalised before being analysed using microscopy, Fluospar Optima readings and FACS analysis.</p><br><br />
<h3>Results</h3><br />
<br />
'''<u>Microscopy</u>'''<br><br />
<p>The microscopy analysis revealed that, in none of the samples, the GFP expression had been completely inhibited. All samples (bar the negative control) showed green fluorescence. The microscope did not allow us to determine if there was any variation however in the levels of GFP in each specific sample. </p><br />
<center><br />
https://static.igem.org/mediawiki/2010/c/c0/MS2vsGFP_fluorescent_cells.jpg<br />
</center><br />
'''<u>Fluospar Optima Readings</u>'''<br><br />
The fluorimeter readings correlated the microscopy results by recording fluorescence in all samples except the – control.<br />
<center><br />
https://static.igem.org/mediawiki/2010/5/5a/MS2vsGFP_fluorimeter.jpg<br />
</center><br />
<p>The recorded fluorescence values for the respective samples showed that there was indeed some variation in the levels of GFP (Fig 1). In both the ‘MS2 Dom’ and the ‘Race’ sample the GFP level was lower than in the + control indicating that the expression of GFP had indeed been inhibited (a 20% decrease for the ‘Race’ sample and an 11% decrease for the ‘MS2’ sample). The ‘GFP Dom’ sample however showed an approximate 8% increase in GFP fluorescence when compared to the + control. Although this is a bit unexpected is could be due to the fact that the GFP expression was initiated in the 1˚ set of conditions whereas it took place in the 2˚ for the + control. However it appears that no inhibition took place indicating that once GFP is being expressed the amount present of MS2 as expressed by MET17p - [MS2] is not able to significantly inhibit the level of GFP fluorescence.</p></div>Josephhttp://2010.igem.org/File:MS2vsGFP_fluorimeter.jpgFile:MS2vsGFP fluorimeter.jpg2010-10-24T12:56:40Z<p>Joseph: </p>
<hr />
<div></div>Josephhttp://2010.igem.org/File:MS2vsGFP_fluorescent_cells.jpgFile:MS2vsGFP fluorescent cells.jpg2010-10-24T12:55:12Z<p>Joseph: </p>
<hr />
<div></div>Josephhttp://2010.igem.org/MS2_Coat-Protein_Effect_on_Expression_of_GFP_in_pRS415MS2 Coat-Protein Effect on Expression of GFP in pRS4152010-10-24T12:54:54Z<p>Joseph: </p>
<hr />
<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<h1> Characterising the translational repression of GAL1p-[Npeptide-GFP] by trans expression of the MS2 protein </h1><br />
<h3>Aim</h3><br />
<p>The characterisation of the effect of MS2 on the expression of GFP by GAL1p-[Npeptide-GFP] will allow more accurate modelling of the system and will allow us to determine with more precision the probability of success of the cross-inhibition of the switch. Expressing MS2 using MET17p - [MS2] will allow us to monitor the effect of MS2 without the complication of the λ-N-peptide produced by GAL1p-[Npeptide-GFP] in turn inhibiting the expression of MS2.</p><br />
<h3>Hypothesis</h3> <br />
<p>The expression of MS2 by MET17p - [MS2]will result in a decrease in the level of expression of GFP by GAL1p-[Npeptide-GFP]. The inhibition will show a linear correlation with the level of expression of MS2.</p><br />
<h3>Protocol</h3><br />
<p>During this experiment double transformants of BY4742 containing GAL1p-[Npeptide-GFP] and MET17p - [MS2] were used. Single transformants of BY4742, containing only GAL1p-[Npeptide-GFP], were used to provide the negative and positive controls for the expression of GFP.</p><br><br />
<p>The double transformants were first cultured overnight in specific conditions in order to establish the desired pre-conditions. The cells were then washed and re-cultured in a different specific set of conditions which would allow the characterisation of the effect of MS2.</p><br><br />
<center><br />
https://static.igem.org/mediawiki/2010/0/04/Conditions_for_FACS_analysis_of_MS2vsGFP.jpg<br />
</center><br />
<p>* 500μM Met was used as this concentration was been used in other experiments to successfully completely switch of the Met17 promoter [1]. <br><br />
<br><br />
The different pre-established conditions allow us to determine whether the history of the sample affects the final result.<br><br />
Final samples were then washed and normalised before being analysed using microscopy, Fluospar Optima readings and FACS analysis.</p><br><br />
<h3>Results</h3><br />
<br />
'''<u>Microscopy</u>'''<br><br />
<p>The microscopy analysis revealed that, in none of the samples, the GFP expression had been completely inhibited. All samples (bar the negative control) showed green fluorescence. The microscope did not allow us to determine if there was any variation however in the levels of GFP in each specific sample. </p></div>Josephhttp://2010.igem.org/File:Conditions_for_FACS_analysis_of_MS2vsGFP.jpgFile:Conditions for FACS analysis of MS2vsGFP.jpg2010-10-24T12:50:08Z<p>Joseph: </p>
<hr />
<div></div>Josephhttp://2010.igem.org/MS2_Coat-Protein_Effect_on_Expression_of_GFP_in_pRS415MS2 Coat-Protein Effect on Expression of GFP in pRS4152010-10-24T12:44:01Z<p>Joseph: </p>
<hr />
<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<h1> Characterising the translational repression of GAL1p-[Npeptide-GFP] by trans expression of the MS2 protein </h1><br />
<h3>Aim</h3><br />
<p>The characterisation of the effect of MS2 on the expression of GFP by GAL1p-[Npeptide-GFP] will allow more accurate modelling of the system and will allow us to determine with more precision the probability of success of the cross-inhibition of the switch. Expressing MS2 using MET17p - [MS2] will allow us to monitor the effect of MS2 without the complication of the λ-N-peptide produced by GAL1p-[Npeptide-GFP] in turn inhibiting the expression of MS2.</p><br />
<h3>Hypothesis</h3> <br />
<p>The expression of MS2 by MET17p - [MS2]will result in a decrease in the level of expression of GFP by GAL1p-[Npeptide-GFP]. The inhibition will show a linear correlation with the level of expression of MS2.</p><br />
<h3>Protocol</h3><br />
<p>During this experiment double transformants of BY4742 containing GAL1p-[Npeptide-GFP] and MET17p - [MS2] were used. Single transformants of BY4742, containing only GAL1p-[Npeptide-GFP], were used to provide the negative and positive controls for the expression of GFP.</p><br><br />
<p>The double transformants were first cultured overnight in specific conditions in order to establish the desired pre-conditions. The cells were then washed and re-cultured in a different specific set of conditions which would allow the characterisation of the effect of MS2.</p><br></div>Josephhttp://2010.igem.org/Team:Aberdeen_Scotland/Switch_CharacterisationTeam:Aberdeen Scotland/Switch Characterisation2010-10-24T11:34:26Z<p>Joseph: </p>
<hr />
<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<br />
<h1> Switch Characterisation Lab-Diary</h1><br><br />
<h2>Prep Week (7th – 11th of June)</h2><br><br />
<ul><li>1.Confirmation of successfully integrated cassettes at the Trp1 locus of:<br><br />
<ul><li>i.N25 with Pgal1 – GFP <br><br />
<li>ii.N4 with Pcup1 – GFP<br></ul><br />
<li>2.Confirmation of GFP expression of N4 and N25 using microscope.<br><br />
<li>3.Promoter characterisation: GFP induction experiment using the fluorometer on:<br><br />
<ul><li>i.BY4741: N25 with Pgal1 transformants<br><br />
<li>ii.BY4741: N4 with Pcup1 transformants<br></ul></ul><br />
<br><br />
<h2>Week1 (14th – 18th of June)</h2><br><br />
<ul><li>1.Transformation of pRS414 and pRS415 into BY4741ΔTrp<br><br />
<li>2.Quantitative determination of the doubling time of BY4741ΔTrp<br><br />
<li>3.Yeast (BY4741) transformation of constructs:<br><br />
<ul><li>i.pRS415<br><br />
<li>ii.pRS414<br></ul></ul><br />
<br><br />
<h2>Week2 (21st – 25th of June)</h2><br><br />
<ul><li>1.Dose response experiment using Pcup (from N4)<br><br />
<li>2.Research for parameter values for theoticians models<br><br />
<li>3.Doubling Rate of N25 grown on SD with Raffinose as a carbon source<br><br />
<li>4.FACS experiment of both Prs414 and prs415, dose response and Time induction set up but experimental and construct errors meant the experiment had to be trouble shooted and then tried again in the future.<br></ul><br />
<br><br />
<h2>Week3 (28th – 2nd July)</h2><br><br />
<ul><li>1.Further research for parameter values for theoticians models.<br><br />
<li>2.Problem solving experiment to determine why no fluorescent expression was observed from the previous FACS. Microscope (using fluorescent filter) observations of fluorescence in:<br><br />
<ul><li>i.N4<br><br />
<li>ii.N25<br><br />
<li>iii.pRS414<br><br />
<li>iv.pRS415<br></ul><br />
<li>3.GFP expression: pRS415 dose response (0.5, 1, 2, 3, 4, 5% of galactose) using the Fluorometer.<br><br />
<li>4.GFP(pRS415) and CFP(pRS414) expression using FACS analysis<br><br />
<li>5.Time induction experiment using Pcup (from N4) <br><br />
<li>6.Transforming pRS414, pRS415 and pMS2 into E.coli (bulking them up)<br></ul><br />
<br><br />
<h2>Week4 (5th – 9th of July)</h2><br><br />
<ul><li>1.Designing primers to modify pMS2 (replacement for pRS414)<br><br />
<li>2.Checking the presence of Age1 in pMS2 / Prep of cut pMS2<br><br />
<li>3.Transformation of pMS2 into BY4742<br><br />
<li>4.Digest of YCp lac 22 Fl<br><br />
<li>5.Time induction experiment of Prs415 on the fluorimeter, no valid results obtained due to sample being frozen overnight.<br></ul><br />
<br><br />
<h2>Week5 (12th – 16th of July)</h2><br><br />
<ul><li>1.Homologous recombination of CFP into YCp lac 22 Fl<br><br />
<li>2.Microscope analysis of YCP-CFP expression<br><br />
<li>3.Made Master plates of BY4742 transformants<br></ul><br />
<br><br />
<h2>Week6 (19th – 23rd of July)</h2><br><br />
<ul><li>1.Time inhibition of GFP expression of pRS415 when inhibitor added on fluorometer.<br><br />
<li>2.Time induction of GFP expression on fluorometer.<br></ul><br />
<br><br />
<h2>Week7 (26th – 30th of July)</h2><br><br />
<ul><li>1.PCR amplification of Bbox and CFP (from pRS414) and GFP (from pRS415)<br><br />
<li>2.Ligated BBox into pMS2 and transformed into E.coli<br><br />
<li>3.PCR of N-GFP homologous sequences<br><br />
<li>4.Transformation of BY4741 ΔTrp with pRS414 and N-GFP (homologous recombination)<br><br />
<li>5.Made master plates of BY4741 pRS414-N-GFP transforms<br></ul><br />
<br><br />
<h2>Week8 (2nd – 7th of August)</h2><br><br />
<ul><li>1.Experiment observing the effect of MS2 (under control of Met17) on expression of GFP (415) [microscopy + fluorspar readings]<br><br />
<li>2.Designing primers for amplifying Cup1-2 (from N4)<br><br />
<li>3.Tested BY4741 pRS414-N-GFP transformants <br></ul><br />
<br><br />
<h2>Week 9 (9th – 14th of August)</h2><br><br />
<ul><li>1.Encountered problems with growing pRS415 for planned FACS induction experiments, thus replaced plates to replenish nutrients, experiments to be repeated week beginning 16th of August.<br><br />
<li>2.FACS analysis of MS2 vs GFP expression<br><br />
<li>3.PCR amplification of CUP promoter form N4 and transformation into prs414<br><br />
<li>4.PCR Colony screening pRS414-N-GFP transformants<br></ul></div>Josephhttp://2010.igem.org/Team:Aberdeen_Scotland/Biobrick_RelatedTeam:Aberdeen Scotland/Biobrick Related2010-10-24T11:28:59Z<p>Joseph: </p>
<hr />
<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<h1>Lab Diary concerning Biobricks</h1><br><br />
<br />
<h2>Preparation week</h2><br><br />
<ul><li>1.Preparation / rescue of iGEM DNA samples:<br><br />
<ul><br />
<li>i.BBa_E2050 – mOrange fluorescent protein, KanR.<br><br />
<li>ii.BBa_J63005 – ADH1 promoter, AmpR.<br><br />
<li>iii.BBa_E2030 – yEYFP (yellow fluorescent protein), KanR.<br><br />
<li>iv.BBa_I716101 with J04450 (plasmid), AmpR.<br><br />
<li>v.BBa_I716101 with P1010 (plasmid with CcdB), AmpR<br></ul><br />
<li>2.Transformation of rescued iGEM DNA samples into XL1 – Blue Subcloning – Grade Competent Cells.<br><br />
<li>3.Transfer of Transformants into fresh plates to prevent the depletion of nutrients.<br></ul><br />
<br><br />
<h2>Week1 (14th – 18th of June)</h2><br><br />
<ul><li>1.Confirmation of the identity of Biobricks YFP (pSB2k3 + E2030)<br><br />
<li>2.Plasmid purification (Miniprep) of desired BioBrick constructs:<br><br />
<ul><li>i.BBa_J63010 plasmid and BBa_J63005 (ADH1 promoter)<br><br />
<li>ii.BBa_I716101 plasmid and BBa_J04450 (RFP)<br><br />
<li>iii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br></ul><br />
<li>3.BioBricks digestion – gel electrophoresis, to verify plasmid sizes, of:<br><br />
<ul><li>i.BBa_J63010 plasmid and BBa_J63005 (ADH1 promoter)<br><br />
<li>ii.BBa_I716101 plasmid and BBa_J04450 (RFP)<br><br />
<li>iii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br></ul></ul><br />
<br><br />
<h2>Week2 (21st – 25th of June)</h2><br><br />
<ul><li>1.Design of Primers for construction of Biobricks<br><br />
<ul><li>i.MS2 protein<br><br />
<li>ii.MS2 loops: 5’leader<br><br />
<li>iii.N peptide (1)<br><br />
<li>iv.N peptide (2)<br><br />
<li>v.Bbox – 5’leader<br></ul><br />
<li>2.Due to unclear results obtained in the prior digestion we repeated the digestion of BioBricks:<br><br />
<ul><li>i.BBa_I716101 plasmid and BBa_J04450 (RFP)<br><br />
<li>ii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br></ul></ul><br />
<br><br />
<h2>Week3 (28th – 2nd July)</h2><br><br />
<ul><li>1.Bgl Brick Preparation:<br><br />
<ul><li>i.Digestion 1, of BglBrick 1, using EcoR1 and Xho1 – higher volume than protocol since concentration of plasmid was very low. <br><br />
<li>ii.Enzymes then heat inactivated<br><br />
<li>iii.Vector then alkaline phosphatase treated, with enzymes again heat inactived after treatment.<br></ul></ul><br />
<br><br />
<h2>Week4 (5th – 9th of July)</h2><br><br />
<ul><li>1.Miniprep of BglBrick 1<br><br />
<li>2.Digestion of BglBrick using EcoR1 and Xho1 – due to low yield when previously digested.<br><br />
<li>3.PCR 1 reaction of inserts (to use for BglBrick cloning)<br><br />
<li>4.Transformation of BglBrick into one shot competent cells E.coli cells, to try and improve plasmid propagation – no improved propagation observed.<br></ul><br />
<br><br />
<h2>Week5 (12th – 16th of July)</h2><br><br />
<ul><li>1.Further Bgl Brick preparation:<br><br />
<ul><li>i.Miniprep of BglBrick<br><br />
<li>ii.Digestion of BglBrick<br><br />
<li>iii.Enzyme heat inactivation<br><br />
<li>iv.Alkaline phosphatase treatment.<br></ul><br />
<li>2.Digestion 1 of PCR products using EcoR1 and Xho1<br><br />
<li>3.Ligation 1 of PCR products (inserts 1) and vector BglBrick<br><br />
<ul><li>Used Nanodrop to determine concentration, we have concluded the data obtained in this way is unreliable, which is why this procedure may not have been successful.<br></ul><br />
<li>4.Transformation 1, of cloned products into XL1 Blue subcloning – grade cells E.coli cells.<br><br />
<li>5.Transformation 2, of cloned products into XL1 Blue competent E.coli cells. Since transformation 1 gave poor efficiency, a more competent cell was tried.<br><br />
<li>6.PCR of E.coli colonies from BglBrick 1 – Did not work.<br><br />
<li>7.Design and order of mOrange primers for Bio-brick testing<br><br />
<li>8.Background reading for Bgl-Brick vector and Bgl-bricking<br><br />
<li>9.Mini-prep, digest and gel electrophoresis of Bgl-Brick vector and pRS415 vector <br></ul><br />
<br><br />
<h2>Week6 (19th – 23rd of July)</h2><br><br />
<ul><li>1.More Bgl Brick preparation since previous involved Nanodrop concentration determination but was decided this analysis is unrealiable:<br><br />
<ul><li>i.Miniprep of BglBrick<br><br />
<li>ii.Digestion of BglBrick<br><br />
<li>iii.Enzyme heat inactivation<br><br />
<li>iv.Alkaline phosphatase treatment.<br></ul><br />
<li>2.Background reading and planning for PCR <br><br />
<li>3.Background reading and planning of alkaline phosphatase and ligation reaction for Bgl-bricking <br></ul><br />
<br><br />
<h2>Week7 (26th – 30th of July)</h2><br><br />
<ul><li>1.PCR of E.coli colonies from BglBrick 2 – positive PCR.<br><br />
<li>2.Ligation 2, of PCR products (inserts 1) and vector BglBrick<br><br />
<li>3.Transformation 3, of cloned products (inserts 1) into subcloning efficiency DH5α E.coli cells.<br><br />
<li>4.PCR of Transformation 3 colonies which were then also plated, to confirm required insert presence.<br><br />
<li>5.Second stage digest , (PvuII) of pRS415 and gel electrophoeris for Bgl-bricking <br><br />
<li>6.Master plate of Bgl-brick<br><br />
<li>7.PCR of mORange<br><br />
<li>8.Transformation of BY4741 ΔTrp with pRS415 and mOrange (homologous recombination)<br></ul><br />
<br><br />
<h2>Week8 (2nd – 7th of August)</h2><br><br />
<ul><li>1.Confirmation of BglBricks produced:<br><br />
<ul><li>i.Plasmid purification (miniprep) of E.coli colonies from Transformation 3 colonies.<br><br />
<li>ii.Digestion of plasmid with<br><br />
<li>iii. Ecor1 and Xhol1<br><br />
<li>iv.Ecor1 – since insert was not being observed on the gel when cut with two enzymes.<br></ul><br />
<li>2.Further Bgl Brick preparation as we ran out:<br><br />
<ul><li>i.6 X Miniprep of BglBrick<br><br />
<li>ii.Combined all 6 from step i. To obtain a higher concentration of BglBrick using Qiagen PCR kit (check name of kit).<br><br />
<li>iii.Digestion of BglBrick using EcoR1 and Xho1<br><br />
<li>iv.Digestion had to be done twice as some uncut vector remained after the first cut.<br><br />
<li>v.Enzyme heat inactivation<br><br />
<li>vi.Alkaline phosphatase treatment.<br></ul><br />
<li>3.Tested BY4741 pRS415 mOrange transformants <br><br />
<li>4.Read through protocol for PCR colony screening <br></ul><br />
<br><br />
<h2>Week 9 (9th – 14th of August)</h2><br><br />
<ul><li>1.Digestion 1, of E.coli colonies from BglBrick 2 using EcoR1 and Xho1.<br><br />
<li>2.Digestion 2, of E.coli colonies from BglBrick 2 using EcoR1, since digest with two enzymes showed no insert band on the gel<br><br />
<li>3.Digestion 2, of PCR products using EcoR1 and Xho1<br><br />
<li>4.Ligation 2, of PCR products (inserts 1) and vector BglBrick<br><br />
<li>5.Transformation 2, of cloned products into XL1 Blue competent – grade E.coli cells.<br><br />
<li>6.PCR Colony screening of mOrange transformants<br><br />
<li>7.Bgl-bricking transformation experiment<br><br />
<li>8.Tested BY4741 pRS415 mOrange transformants using fluorometer<br><br />
<li>9.Mini-prep Bgl-brick vectors<br></ul></div>Josephhttp://2010.igem.org/Team:Aberdeen_Scotland/Biobrick_RelatedTeam:Aberdeen Scotland/Biobrick Related2010-10-24T11:26:15Z<p>Joseph: </p>
<hr />
<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<h1>Lab Diary concerning Biobricks</h1><br><br />
<br />
<h2>Preparation week</h2><br><br />
<ul><li>1.Preparation / rescue of iGEM DNA samples:<br><br />
<ul><br />
<li>i.BBa_E2050 – mOrange fluorescent protein, KanR.<br><br />
<li>ii.BBa_J63005 – ADH1 promoter, AmpR.<br><br />
<li>iii.BBa_E2030 – yEYFP (yellow fluorescent protein), KanR.<br><br />
<li>iv.BBa_I716101 with J04450 (plasmid), AmpR.<br><br />
<li>v.BBa_I716101 with P1010 (plasmid with CcdB), AmpR<br></ul><br />
<li>2.Transformation of rescued iGEM DNA samples into XL1 – Blue Subcloning – Grade Competent Cells.<br><br />
<li>3.Transfer of Transformants into fresh plates to prevent the depletion of nutrients.<br></ul><br />
<br><br />
<h2>Week1 (14th – 18th of June)</h2><br><br />
<ul><li>1.Confirmation of the identity of Biobricks YFP (pSB2k3 + E2030)<br><br />
<li>2.Plasmid purification (Miniprep) of desired BioBrick constructs:<br><br />
<ul><li>i.BBa_J63010 plasmid and BBa_J63005 (ADH1 promoter)<br><br />
<li>ii.BBa_I716101 plasmid and BBa_J04450 (RFP)<br><br />
<li>iii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br></ul><br />
<li>3.BioBricks digestion – gel electrophoresis, to verify plasmid sizes, of:<br><br />
<ul><li>i.BBa_J63010 plasmid and BBa_J63005 (ADH1 promoter)<br><br />
<li>ii.BBa_I716101 plasmid and BBa_J04450 (RFP)<br><br />
<li>iii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br></ul></ul><br />
<br><br />
<h2>Week2 (21st – 25th of June)</h2><br><br />
<ul><li>1.Design of Primers for construction of Biobricks<br><br />
<ul><li>i.MS2 protein<br><br />
<li>ii.MS2 loops: 5’leader<br><br />
<li>iii.N peptide (1)<br><br />
<li>iv.N peptide (2)<br><br />
<li>v.Bbox – 5’leader<br></ul><br />
<li>2.Due to unclear results obtained in the prior digestion we repeated the digestion of BioBricks:<br><br />
<ul><li>i.BBa_I716101 plasmid and BBa_J04450 (RFP)<br><br />
<li>ii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br></ul></ul><br />
<br><br />
<h2>Week3 (28th – 2nd July)</h2><br><br />
<ul><li>1.Bgl Brick Preparation:<br><br />
<ul><li>i.Digestion 1, of BglBrick 1, using EcoR1 and Xho1 – higher volume than protocol since concentration of plasmid was very low. <br><br />
<li>ii.Enzymes then heat inactivated<br><br />
<li>iii.Vector then alkaline phosphatase treated, with enzymes again heat inactived after treatment.<br></ul></ul><br />
<br><br />
<h2>Week4 (5th – 9th of July)</h2><br><br />
<ul><li>1.Miniprep of BglBrick 1<br><br />
<li>2.Digestion of BglBrick using EcoR1 and Xho1 – due to low yield when previously digested.<br><br />
<li>3.PCR 1 reaction of inserts (to use for BglBrick cloning)<br><br />
<li>4.Transformation of BglBrick into one shot competent cells E.coli cells, to try and improve plasmid propagation – no improved propagation observed.<br></ul><br />
<br><br />
<h2>Week5 (12th – 16th of July)</h2><br><br />
<ul><li>1.Further Bgl Brick preparation:<br><br />
<ul><li>i.Miniprep of BglBrick<br><br />
<li>ii.Digestion of BglBrick<br><br />
<li>iii.Enzyme heat inactivation<br><br />
<li>iv.Alkaline phosphatase treatment.<br></ul><br />
<li>2.Digestion 1 of PCR products using EcoR1 and Xho1<br><br />
<li>3.Ligation 1 of PCR products (inserts 1) and vector BglBrick<br><br />
<ul><li>Used Nanodrop to determine concentration, we have concluded the data obtained in this way is unreliable, which is why this procedure may not have been successful.<br></ul><br />
<li>4.Transformation 1, of cloned products into XL1 Blue subcloning – grade cells E.coli cells.<br><br />
<li>5.Transformation 2, of cloned products into XL1 Blue competent E.coli cells. Since transformation 1 gave poor efficiency, a more competent cell was tried.<br><br />
<li>6.PCR of E.coli colonies from BglBrick 1 – Did not work.<br><br />
<li>7.Design and order of mOrange primers for Bio-brick testing<br><br />
<li>8.Background reading for Bgl-Brick vector and Bgl-bricking<br><br />
<li>9.Mini-prep, digest and gel electrophoresis of Bgl-Brick vector and pRS415 vector <br></ul><br />
<br><br />
<h2>Week6 (19th – 23rd of July)</h2><br><br />
<ul><li>1.More Bgl Brick preparation since previous involved Nanodrop concentration determination but was decided this analysis is unrealiable:<br><br />
<ul><li>i.Miniprep of BglBrick<br><br />
<li>ii.Digestion of BglBrick<br><br />
<li>iii.Enzyme heat inactivation<br><br />
<li>iv.Alkaline phosphatase treatment.<br></ul><br />
<li>2.Background reading and planning for PCR <br><br />
<li>3.Background reading and planning of alkaline phosphatase and ligation reaction for Bgl-bricking <br></ul><br />
<br><br />
<h2>Week7 (26th – 30th of July)</h2><br><br />
1.PCR of E.coli colonies from BglBrick 2 – positive PCR.<br><br />
2.Ligation 2, of PCR products (inserts 1) and vector BglBrick<br><br />
3.Transformation 3, of cloned products (inserts 1) into subcloning efficiency DH5α E.coli cells.<br><br />
4.PCR of Transformation 3 colonies which were then also plated, to confirm required insert presence.<br><br />
5.Second stage digest , (PvuII) of pRS415 and gel electrophoeris for Bgl-bricking <br><br />
6.Master plate of Bgl-brick<br><br />
7.PCR of mORange<br><br />
8.Transformation of BY4741 ΔTrp with pRS415 and mOrange (homologous recombination)<br><br />
<br><br />
<h2>Week8 (2nd – 7th of August)</h2><br><br />
1.Confirmation of BglBricks produced:<br><br />
i.Plasmid purification (miniprep) of E.coli colonies from Transformation 3 colonies.<br><br />
ii.Digestion of plasmid with<br><br />
iii. Ecor1 and Xhol1<br><br />
iv.Ecor1 – since insert was not being observed on the gel when cut with two enzymes.<br><br />
2.Further Bgl Brick preparation as we ran out:<br><br />
i.6 X Miniprep of BglBrick<br><br />
ii.Combined all 6 from step i. To obtain a higher concentration of BglBrick using Qiagen PCR kit (check name of kit).<br><br />
iii.Digestion of BglBrick using EcoR1 and Xho1<br><br />
iv.Digestion had to be done twice as some uncut vector remained after the first cut.<br><br />
v.Enzyme heat inactivation<br><br />
vi.Alkaline phosphatase treatment.<br><br />
3.Tested BY4741 pRS415 mOrange transformants <br><br />
4.Read through protocol for PCR colony screening <br><br />
<br><br />
<h2>Week 9 (9th – 14th of August)</h2><br><br />
1.Digestion 1, of E.coli colonies from BglBrick 2 using EcoR1 and Xho1.<br><br />
2.Digestion 2, of E.coli colonies from BglBrick 2 using EcoR1, since digest with two enzymes showed no insert band on the gel<br><br />
3.Digestion 2, of PCR products using EcoR1 and Xho1<br><br />
4.Ligation 2, of PCR products (inserts 1) and vector BglBrick<br><br />
5.Transformation 2, of cloned products into XL1 Blue competent – grade E.coli cells.<br><br />
6.PCR Colony screening of mOrange transformants<br><br />
7.Bgl-bricking transformation experiment<br><br />
8.Tested BY4741 pRS415 mOrange transformants using fluorometer<br><br />
9.Mini-prep Bgl-brick vectors<br></div>Josephhttp://2010.igem.org/Team:Aberdeen_Scotland/Biobrick_RelatedTeam:Aberdeen Scotland/Biobrick Related2010-10-24T11:19:02Z<p>Joseph: </p>
<hr />
<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<h1>Lab Diary concerning Biobricks</h1><br><br />
<br />
<h2>Preparation week</h2><br><br />
<ul><li>1.Preparation / rescue of iGEM DNA samples:<br><br />
<ul><br />
<li>i.BBa_E2050 – mOrange fluorescent protein, KanR.<br><br />
<li>ii.BBa_J63005 – ADH1 promoter, AmpR.<br><br />
<li>iii.BBa_E2030 – yEYFP (yellow fluorescent protein), KanR.<br><br />
<li>iv.BBa_I716101 with J04450 (plasmid), AmpR.<br><br />
<li>v.BBa_I716101 with P1010 (plasmid with CcdB), AmpR<br></ul><br />
<li>2.Transformation of rescued iGEM DNA samples into XL1 – Blue Subcloning – Grade Competent Cells.<br><br />
<li>3.Transfer of Transformants into fresh plates to prevent the depletion of nutrients.<br></ul><br />
<br><br />
<h2>Week1 (14th – 18th of June)</h2><br><br />
<ul><li>1.Confirmation of the identity of Biobricks YFP (pSB2k3 + E2030)<br><br />
<li>2.Plasmid purification (Miniprep) of desired BioBrick constructs:<br><br />
<ul><li>i.BBa_J63010 plasmid and BBa_J63005 (ADH1 promoter)<br><br />
<li>ii.BBa_I716101 plasmid and BBa_J04450 (RFP)<br><br />
<li>iii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br></ul><br />
<li>3.BioBricks digestion – gel electrophoresis, to verify plasmid sizes, of:<br><br />
<ul><li>i.BBa_J63010 plasmid and BBa_J63005 (ADH1 promoter)<br><br />
<li>ii.BBa_I716101 plasmid and BBa_J04450 (RFP)<br><br />
<li>iii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br></ul></ul><br />
<br><br />
<h2>Week2 (21st – 25th of June)</h2><br><br />
1.Design of Primers for construction of Biobricks<br><br />
i.MS2 protein<br><br />
ii.MS2 loops: 5’leader<br><br />
iii.N peptide (1)<br><br />
iv.N peptide (2)<br><br />
v.Bbox – 5’leader<br><br />
2.Due to unclear results obtained in the prior digestion we repeated the digestion of BioBricks:<br><br />
i.BBa_I716101 plasmid and BBa_J04450 (RFP)<br><br />
ii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br><br />
<br><br />
<h2>Week3 (28th – 2nd July)</h2><br><br />
1.Bgl Brick Preparation:<br><br />
i.Digestion 1, of BglBrick 1, using EcoR1 and Xho1 – higher volume than protocol since concentration of plasmid was very low. <br><br />
ii.Enzymes then heat inactivated<br><br />
iii.Vector then alkaline phosphatase treated, with enzymes again heat inactived after treatment.<br><br />
<br><br />
<h2>Week4 (5th – 9th of July)</h2><br><br />
1.Miniprep of BglBrick 1<br><br />
2.Digestion of BglBrick using EcoR1 and Xho1 – due to low yield when previously digested.<br><br />
3.PCR 1 reaction of inserts (to use for BglBrick cloning)<br><br />
4.Transformation of BglBrick into one shot competent cells E.coli cells, to try and improve plasmid propagation – no improved propagation observed.<br><br />
<br><br />
<h2>Week5 (12th – 16th of July)</h2><br><br />
1.Further Bgl Brick preparation:<br><br />
i.Miniprep of BglBrick<br><br />
ii.Digestion of BglBrick<br><br />
iii.Enzyme heat inactivation<br><br />
iv.Alkaline phosphatase treatment.<br><br />
2.Digestion 1 of PCR products using EcoR1 and Xho1<br><br />
3.Ligation 1 of PCR products (inserts 1) and vector BglBrick<br><br />
i.Used Nanodrop to determine concentration, we have concluded the data obtained in this way is unreliable, which is why this procedure may not have been successful.<br><br />
4.Transformation 1, of cloned products into XL1 Blue subcloning – grade cells E.coli cells.<br><br />
5.Transformation 2, of cloned products into XL1 Blue competent E.coli cells. Since transformation 1 gave poor efficiency, a more competent cell was tried.<br><br />
6.PCR of E.coli colonies from BglBrick 1 – Did not work.<br><br />
7.Design and order of mOrange primers for Bio-brick testing<br><br />
8.Background reading for Bgl-Brick vector and Bgl-bricking<br><br />
9.Mini-prep, digest and gel electrophoresis of Bgl-Brick vector and pRS415 vector <br><br />
<br><br />
<h2>Week6 (19th – 23rd of July)</h2><br><br />
1.More Bgl Brick preparation since previous involved Nanodrop concentration determination but was decided this analysis is unrealiable:<br><br />
i.Miniprep of BglBrick<br><br />
ii.Digestion of BglBrick<br><br />
iii.Enzyme heat inactivation<br><br />
iv.Alkaline phosphatase treatment.<br><br />
2.Background reading and planning for PCR <br><br />
3.Background reading and planning of alkaline phosphatase and ligation reaction for Bgl-bricking <br><br />
<br><br />
<h2>Week7 (26th – 30th of July)</h2><br><br />
1.PCR of E.coli colonies from BglBrick 2 – positive PCR.<br><br />
2.Ligation 2, of PCR products (inserts 1) and vector BglBrick<br><br />
3.Transformation 3, of cloned products (inserts 1) into subcloning efficiency DH5α E.coli cells.<br><br />
4.PCR of Transformation 3 colonies which were then also plated, to confirm required insert presence.<br><br />
5.Second stage digest , (PvuII) of pRS415 and gel electrophoeris for Bgl-bricking <br><br />
6.Master plate of Bgl-brick<br><br />
7.PCR of mORange<br><br />
8.Transformation of BY4741 ΔTrp with pRS415 and mOrange (homologous recombination)<br><br />
<br><br />
<h2>Week8 (2nd – 7th of August)</h2><br><br />
1.Confirmation of BglBricks produced:<br><br />
i.Plasmid purification (miniprep) of E.coli colonies from Transformation 3 colonies.<br><br />
ii.Digestion of plasmid with<br><br />
iii. Ecor1 and Xhol1<br><br />
iv.Ecor1 – since insert was not being observed on the gel when cut with two enzymes.<br><br />
2.Further Bgl Brick preparation as we ran out:<br><br />
i.6 X Miniprep of BglBrick<br><br />
ii.Combined all 6 from step i. To obtain a higher concentration of BglBrick using Qiagen PCR kit (check name of kit).<br><br />
iii.Digestion of BglBrick using EcoR1 and Xho1<br><br />
iv.Digestion had to be done twice as some uncut vector remained after the first cut.<br><br />
v.Enzyme heat inactivation<br><br />
vi.Alkaline phosphatase treatment.<br><br />
3.Tested BY4741 pRS415 mOrange transformants <br><br />
4.Read through protocol for PCR colony screening <br><br />
<br><br />
<h2>Week 9 (9th – 14th of August)</h2><br><br />
1.Digestion 1, of E.coli colonies from BglBrick 2 using EcoR1 and Xho1.<br><br />
2.Digestion 2, of E.coli colonies from BglBrick 2 using EcoR1, since digest with two enzymes showed no insert band on the gel<br><br />
3.Digestion 2, of PCR products using EcoR1 and Xho1<br><br />
4.Ligation 2, of PCR products (inserts 1) and vector BglBrick<br><br />
5.Transformation 2, of cloned products into XL1 Blue competent – grade E.coli cells.<br><br />
6.PCR Colony screening of mOrange transformants<br><br />
7.Bgl-bricking transformation experiment<br><br />
8.Tested BY4741 pRS415 mOrange transformants using fluorometer<br><br />
9.Mini-prep Bgl-brick vectors<br></div>Josephhttp://2010.igem.org/Team:Aberdeen_Scotland/Biobrick_RelatedTeam:Aberdeen Scotland/Biobrick Related2010-10-24T11:05:32Z<p>Joseph: </p>
<hr />
<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<h1>Lab Diary concerning Biobricks</h1><br><br />
<br />
<h2>Preparation week</h2><br><br />
1.Preparation / rescue of iGEM DNA samples:<br><br />
i.BBa_E2050 – mOrange fluorescent protein, KanR.<br><br />
ii.BBa_J63005 – ADH1 promoter, AmpR.<br><br />
iii.BBa_E2030 – yEYFP (yellow fluorescent protein), KanR.<br><br />
iv.BBa_I716101 with J04450 (plasmid), AmpR.<br><br />
v.BBa_I716101 with P1010 (plasmid with CcdB), AmpR<br><br />
2.Transformation of rescued iGEM DNA samples into XL1 – Blue Subcloning – Grade Competent Cells.<br><br />
3.Transfer of Transformants into fresh plates to prevent the depletion of nutrients.<br><br />
<br><br />
<h2>Week1 (14th – 18th of June)</h2><br><br />
1.Confirmation of the identity of Biobricks YFP (pSB2k3 + E2030)<br><br />
2.Plasmid purification (Miniprep) of desired BioBrick constructs:<br><br />
i.BBa_J63010 plasmid and BBa_J63005 (ADH1 promoter)<br><br />
ii.BBa_I716101 plasmid and BBa_J04450 (RFP)<br><br />
iii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br><br />
3.BioBricks digestion – gel electrophoresis, to verify plasmid sizes, of:<br><br />
i.BBa_J63010 plasmid and BBa_J63005 (ADH1 promoter)<br><br />
ii.BBa_I716101 plasmid and BBa_J04450 (RFP)<br><br />
iii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br><br />
<br><br />
<h2>Week2 (21st – 25th of June)</h2><br><br />
1.Design of Primers for construction of Biobricks<br><br />
i.MS2 protein<br><br />
ii.MS2 loops: 5’leader<br><br />
iii.N peptide (1)<br><br />
iv.N peptide (2)<br><br />
v.Bbox – 5’leader<br><br />
2.Due to unclear results obtained in the prior digestion we repeated the digestion of BioBricks:<br><br />
i.BBa_I716101 plasmid and BBa_J04450 (RFP)<br><br />
ii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br><br />
<br><br />
<h2>Week3 (28th – 2nd July)</h2><br><br />
1.Bgl Brick Preparation:<br><br />
i.Digestion 1, of BglBrick 1, using EcoR1 and Xho1 – higher volume than protocol since concentration of plasmid was very low. <br><br />
ii.Enzymes then heat inactivated<br><br />
iii.Vector then alkaline phosphatase treated, with enzymes again heat inactived after treatment.<br><br />
<br><br />
<h2>Week4 (5th – 9th of July)</h2><br><br />
1.Miniprep of BglBrick 1<br><br />
2.Digestion of BglBrick using EcoR1 and Xho1 – due to low yield when previously digested.<br><br />
3.PCR 1 reaction of inserts (to use for BglBrick cloning)<br><br />
4.Transformation of BglBrick into one shot competent cells E.coli cells, to try and improve plasmid propagation – no improved propagation observed.<br><br />
<br><br />
<h2>Week5 (12th – 16th of July)</h2><br><br />
1.Further Bgl Brick preparation:<br><br />
i.Miniprep of BglBrick<br><br />
ii.Digestion of BglBrick<br><br />
iii.Enzyme heat inactivation<br><br />
iv.Alkaline phosphatase treatment.<br><br />
2.Digestion 1 of PCR products using EcoR1 and Xho1<br><br />
3.Ligation 1 of PCR products (inserts 1) and vector BglBrick<br><br />
i.Used Nanodrop to determine concentration, we have concluded the data obtained in this way is unreliable, which is why this procedure may not have been successful.<br><br />
4.Transformation 1, of cloned products into XL1 Blue subcloning – grade cells E.coli cells.<br><br />
5.Transformation 2, of cloned products into XL1 Blue competent E.coli cells. Since transformation 1 gave poor efficiency, a more competent cell was tried.<br><br />
6.PCR of E.coli colonies from BglBrick 1 – Did not work.<br><br />
7.Design and order of mOrange primers for Bio-brick testing<br><br />
8.Background reading for Bgl-Brick vector and Bgl-bricking<br><br />
9.Mini-prep, digest and gel electrophoresis of Bgl-Brick vector and pRS415 vector <br><br />
<br><br />
<h2>Week6 (19th – 23rd of July)</h2><br><br />
1.More Bgl Brick preparation since previous involved Nanodrop concentration determination but was decided this analysis is unrealiable:<br><br />
i.Miniprep of BglBrick<br><br />
ii.Digestion of BglBrick<br><br />
iii.Enzyme heat inactivation<br><br />
iv.Alkaline phosphatase treatment.<br><br />
2.Background reading and planning for PCR <br><br />
3.Background reading and planning of alkaline phosphatase and ligation reaction for Bgl-bricking <br><br />
<br><br />
<h2>Week7 (26th – 30th of July)</h2><br><br />
1.PCR of E.coli colonies from BglBrick 2 – positive PCR.<br><br />
2.Ligation 2, of PCR products (inserts 1) and vector BglBrick<br><br />
3.Transformation 3, of cloned products (inserts 1) into subcloning efficiency DH5α E.coli cells.<br><br />
4.PCR of Transformation 3 colonies which were then also plated, to confirm required insert presence.<br><br />
5.Second stage digest , (PvuII) of pRS415 and gel electrophoeris for Bgl-bricking <br><br />
6.Master plate of Bgl-brick<br><br />
7.PCR of mORange<br><br />
8.Transformation of BY4741 ΔTrp with pRS415 and mOrange (homologous recombination)<br><br />
<br><br />
<h2>Week8 (2nd – 7th of August)</h2><br><br />
1.Confirmation of BglBricks produced:<br><br />
i.Plasmid purification (miniprep) of E.coli colonies from Transformation 3 colonies.<br><br />
ii.Digestion of plasmid with<br><br />
iii. Ecor1 and Xhol1<br><br />
iv.Ecor1 – since insert was not being observed on the gel when cut with two enzymes.<br><br />
2.Further Bgl Brick preparation as we ran out:<br><br />
i.6 X Miniprep of BglBrick<br><br />
ii.Combined all 6 from step i. To obtain a higher concentration of BglBrick using Qiagen PCR kit (check name of kit).<br><br />
iii.Digestion of BglBrick using EcoR1 and Xho1<br><br />
iv.Digestion had to be done twice as some uncut vector remained after the first cut.<br><br />
v.Enzyme heat inactivation<br><br />
vi.Alkaline phosphatase treatment.<br><br />
3.Tested BY4741 pRS415 mOrange transformants <br><br />
4.Read through protocol for PCR colony screening <br><br />
<br><br />
<h2>Week 9 (9th – 14th of August)</h2><br><br />
1.Digestion 1, of E.coli colonies from BglBrick 2 using EcoR1 and Xho1.<br><br />
2.Digestion 2, of E.coli colonies from BglBrick 2 using EcoR1, since digest with two enzymes showed no insert band on the gel<br><br />
3.Digestion 2, of PCR products using EcoR1 and Xho1<br><br />
4.Ligation 2, of PCR products (inserts 1) and vector BglBrick<br><br />
5.Transformation 2, of cloned products into XL1 Blue competent – grade E.coli cells.<br><br />
6.PCR Colony screening of mOrange transformants<br><br />
7.Bgl-bricking transformation experiment<br><br />
8.Tested BY4741 pRS415 mOrange transformants using fluorometer<br><br />
9.Mini-prep Bgl-brick vectors<br></div>Josephhttp://2010.igem.org/Team:Aberdeen_Scotland/Switch_CharacterisationTeam:Aberdeen Scotland/Switch Characterisation2010-10-24T10:52:49Z<p>Joseph: </p>
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<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<br />
<h1> Switch Characterisation Lab-Diary</h1><br><br />
<h2>Prep Week (7th – 11th of June)</h2><br><br />
1.Confirmation of successfully integrated cassettes at the Trp1 locus of:<br><br />
i.N25 with Pgal1 – GFP <br><br />
ii.N4 with Pcup1 – GFP<br><br />
2.Confirmation of GFP expression of N4 and N25 using microscope.<br><br />
3.Promoter characterisation: GFP induction experiment using the fluorometer on:<br><br />
i.BY4741: N25 with Pgal1 transformants<br><br />
ii.BY4741: N4 with Pcup1 transformants<br><br />
<br><br />
<h2>Week1 (14th – 18th of June)</h2><br><br />
1.Transformation of pRS414 and pRS415 into BY4741ΔTrp<br><br />
2.Quantitative determination of the doubling time of BY4741ΔTrp<br><br />
3.Yeast (BY4741) transformation of constructs:<br><br />
i.pRS415<br><br />
ii.pRS414<br><br />
<br><br />
<h2>Week2 (21st – 25th of June)</h2><br><br />
1.Dose response experiment using Pcup (from N4)<br><br />
2.Research for parameter values for theoticians models<br><br />
3.Doubling Rate of N25 grown on SD with Raffinose as a carbon source<br><br />
4.FACS experiment of both Prs414 and prs415, dose response and Time induction set up but experimental and construct errors meant the experiment had to be trouble shooted and then tried again in the future.<br><br />
<br><br />
<h2>Week3 (28th – 2nd July)</h2><br><br />
1.Further research for parameter values for theoticians models.<br><br />
2.Problem solving experiment to determine why no fluorescent expression was observed from the previous FACS. Microscope (using fluorescent filter) observations of fluorescence in:<br><br />
i.N4<br><br />
ii.N25<br><br />
iii.pRS414<br><br />
iv.pRS415<br><br />
3.GFP expression: pRS415 dose response (0.5, 1, 2, 3, 4, 5% of galactose) using the Fluorometer.<br><br />
4.GFP(pRS415) and CFP(pRS414) expression using FACS analysis<br><br />
5.Time induction experiment using Pcup (from N4) <br><br />
6.Transforming pRS414, pRS415 and pMS2 into E.coli (bulking them up)<br><br />
<br><br />
<h2>Week4 (5th – 9th of July)</h2><br><br />
1.Designing primers to modify pMS2 (replacement for pRS414)<br><br />
2.Checking the presence of Age1 in pMS2 / Prep of cut pMS2<br><br />
3.Transformation of pMS2 into BY4742<br><br />
4.Digest of YCp lac 22 Fl<br><br />
5.Time induction experiment of Prs415 on the fluorimeter, no valid results obtained due to sample being frozen overnight.<br><br />
<br><br />
<h2>Week5 (12th – 16th of July)</h2><br><br />
1.Homologous recombination of CFP into YCp lac 22 Fl<br><br />
2.Microscope analysis of YCP-CFP expression<br><br />
3.Made Master plates of BY4742 transformants<br><br />
<br><br />
<h2>Week6 (19th – 23rd of July)</h2><br><br />
1.Time inhibition of GFP expression of pRS415 when inhibitor added on fluorometer.<br><br />
2.Time induction of GFP expression on fluorometer.<br><br />
<br><br />
<h2>Week7 (26th – 30th of July)</h2><br><br />
1.PCR amplification of Bbox and CFP (from pRS414) and GFP (from pRS415)<br><br />
2.Ligated BBox into pMS2 and transformed into E.coli<br><br />
3.PCR of N-GFP homologous sequences<br><br />
4.Transformation of BY4741 ΔTrp with pRS414 and N-GFP (homologous recombination)<br><br />
5.Made master plates of BY4741 pRS414-N-GFP transforms<br><br />
<br><br />
<h2>Week8 (2nd – 7th of August)</h2><br><br />
1.Experiment observing the effect of MS2 (under control of Met17) on expression of GFP (415) [microscopy + fluorspar readings]<br><br />
2.Designing primers for amplifying Cup1-2 (from N4)<br><br />
3.Tested BY4741 pRS414-N-GFP transformants <br><br />
<br><br />
<h2>Week 9 (9th – 14th of August)</h2><br><br />
1.Encountered problems with growing pRS415 for planned FACS induction experiments, thus replaced plates to replenish nutrients, experiments to be repeated week beginning 16th of August.<br><br />
2.FACS analysis of MS2 vs GFP expression<br><br />
3.PCR amplification of CUP promoter form N4 and transformation into prs414<br><br />
4.PCR Colony screening pRS414-N-GFP transformants<br></div>Josephhttp://2010.igem.org/Switch_CharacterisationSwitch Characterisation2010-10-24T10:50:43Z<p>Joseph: Removing all content from page</p>
<hr />
<div></div>Josephhttp://2010.igem.org/Switch_CharacterisationSwitch Characterisation2010-10-24T10:49:39Z<p>Joseph: New page: {{:Team:Aberdeen_Scotland/css}} {{:Team:Aberdeen_Scotland/Title}} <h2>Prep Week (7th – 11th of June)</h2><br> 1.Confirmation of successfully integrated cassettes at the Trp1 locus of:<b...</p>
<hr />
<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<br />
<h2>Prep Week (7th – 11th of June)</h2><br><br />
1.Confirmation of successfully integrated cassettes at the Trp1 locus of:<br><br />
i.N25 with Pgal1 – GFP <br><br />
ii.N4 with Pcup1 – GFP<br><br />
2.Confirmation of GFP expression of N4 and N25 using microscope.<br><br />
3.Promoter characterisation: GFP induction experiment using the fluorometer on:<br><br />
i.BY4741: N25 with Pgal1 transformants<br><br />
ii.BY4741: N4 with Pcup1 transformants<br><br />
<br><br />
<h2>Week1 (14th – 18th of June)</h2><br><br />
1.Transformation of pRS414 and pRS415 into BY4741ΔTrp<br><br />
2.Quantitative determination of the doubling time of BY4741ΔTrp<br><br />
3.Yeast (BY4741) transformation of constructs:<br><br />
i.pRS415<br><br />
ii.pRS414<br><br />
<br><br />
<h2>Week2 (21st – 25th of June)</h2><br><br />
1.Dose response experiment using Pcup (from N4)<br><br />
2.Research for parameter values for theoticians models<br><br />
3.Doubling Rate of N25 grown on SD with Raffinose as a carbon source<br><br />
4.FACS experiment of both Prs414 and prs415, dose response and Time induction set up but experimental and construct errors meant the experiment had to be trouble shooted and then tried again in the future.<br><br />
<br><br />
<h2>Week3 (28th – 2nd July)</h2><br><br />
1.Further research for parameter values for theoticians models.<br><br />
2.Problem solving experiment to determine why no fluorescent expression was observed from the previous FACS. Microscope (using fluorescent filter) observations of fluorescence in:<br><br />
i.N4<br><br />
ii.N25<br><br />
iii.pRS414<br><br />
iv.pRS415<br><br />
3.GFP expression: pRS415 dose response (0.5, 1, 2, 3, 4, 5% of galactose) using the Fluorometer.<br><br />
4.GFP(pRS415) and CFP(pRS414) expression using FACS analysis<br><br />
5.Time induction experiment using Pcup (from N4) <br><br />
6.Transforming pRS414, pRS415 and pMS2 into E.coli (bulking them up)<br><br />
<br><br />
<h2>Week4 (5th – 9th of July)</h2><br><br />
1.Designing primers to modify pMS2 (replacement for pRS414)<br><br />
2.Checking the presence of Age1 in pMS2 / Prep of cut pMS2<br><br />
3.Transformation of pMS2 into BY4742<br><br />
4.Digest of YCp lac 22 Fl<br><br />
5.Time induction experiment of Prs415 on the fluorimeter, no valid results obtained due to sample being frozen overnight.<br><br />
<br><br />
<h2>Week5 (12th – 16th of July)</h2><br><br />
1.Homologous recombination of CFP into YCp lac 22 Fl<br><br />
2.Microscope analysis of YCP-CFP expression<br><br />
3.Made Master plates of BY4742 transformants<br><br />
<br><br />
<h2>Week6 (19th – 23rd of July)</h2><br><br />
1.Time inhibition of GFP expression of pRS415 when inhibitor added on fluorometer.<br><br />
2.Time induction of GFP expression on fluorometer.<br><br />
<br><br />
<h2>Week7 (26th – 30th of July)</h2><br><br />
1.PCR amplification of Bbox and CFP (from pRS414) and GFP (from pRS415)<br><br />
2.Ligated BBox into pMS2 and transformed into E.coli<br><br />
3.PCR of N-GFP homologous sequences<br><br />
4.Transformation of BY4741 ΔTrp with pRS414 and N-GFP (homologous recombination)<br><br />
5.Made master plates of BY4741 pRS414-N-GFP transforms<br><br />
<br><br />
<h2>Week8 (2nd – 7th of August)</h2><br><br />
1.Experiment observing the effect of MS2 (under control of Met17) on expression of GFP (415) [microscopy + fluorspar readings]<br><br />
2.Designing primers for amplifying Cup1-2 (from N4)<br><br />
3.Tested BY4741 pRS414-N-GFP transformants <br><br />
<br><br />
<h2>Week 9 (9th – 14th of August)</h2><br><br />
1.Encountered problems with growing pRS415 for planned FACS induction experiments, thus replaced plates to replenish nutrients, experiments to be repeated week beginning 16th of August.<br><br />
2.FACS analysis of MS2 vs GFP expression<br><br />
3.PCR amplification of CUP promoter form N4 and transformation into prs414<br><br />
4.PCR Colony screening pRS414-N-GFP transformants<br></div>Josephhttp://2010.igem.org/Team:Aberdeen_Scotland/HeaderTeam:Aberdeen Scotland/Header2010-10-24T10:26:42Z<p>Joseph: </p>
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</html></div>Josephhttp://2010.igem.org/MS2_coat-protein_effect_on_expression_of_GFP_in_pRS415MS2 coat-protein effect on expression of GFP in pRS4152010-10-08T18:56:35Z<p>Joseph: </p>
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<h1>Characterisation of the inhibition of GFP expression in pRS415 through the binding of the MS2 coat protein to MS2 stem loops</h1><br />
<h3>Aim</h3><br />
<p>The characterisation of the effect of MS2 on the expression of GFP by pRS415 will allow more accurate modelling of the system and will allow us to determine with more precision the probability of success of the cross-inhibition of the switch. Expressing MS2 using the Met17-MS2 vector will allow us to monitor the effect of MS2 without the complication of the λ-N-peptide produced by pRS415 in turn inhibiting the expression of MS2.</p><br />
<h3>Hypothesis</h3><br />
<p>The expression of MS2 by Met17-MS2 will result in a decrease in the level of expression of GFP by pRS415. The inhibition will show a linear correlation with the level of expression of MS2.<br />
<h3>Protocol</h3><br />
<p>During this experiment double transformants of BY4742 containing pRS415 and Met17-MS2 were used. Single transformants of BY4742, containing only pRS415, were used to provide the negative and positive controls for the expression of GFP.<br><br />
<br><br />
The double transformants were first cultured overnight in specific conditions in order to establish the desired pre-conditions. The cells were then washed and re-cultured in a different specific set of conditions which would allow the characterisation of the effect of MS2.<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/e/e5/Setup_for_MS2_charac.jpg"/><br />
</center><br />
<p>The different pre-established conditions allow us to determine whether the history of the sample affects the final result.<br><br />
<br><br />
Final samples were then washed and normalised before being analysed using microscopy, Fluospar Optima readings and FACS analysis.</p><br />
<h3>Results</h3><br />
<h3>Microscopy</h3><br />
<p>The microscopy analysis revealed that, in none of the samples, the GFP expression had been completely inhibited. All samples (bar the negative control) showed green fluorescence. The microscope did not allow us to determine if there was any variation however in the levels of GFP in each specific sample.</p> <br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/b1/GFP_fluo_under_MS2_repression.jpg"/><br />
</center><br />
<h3>Fluospar Optima Readings</h3><br />
<p>The fluorimeter readings correlated the microscopy results by recording fluorescence in all samples except the – control.</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/d/d2/Fluorimeter_graph_for_Ms2_repression.jpg"/><br />
</center><br />
<p>The recorded fluorescence values for the respective samples showed that there was indeed some variation in the levels of GFP (Fig 1). In both the ‘MS2 Dom’ and the ‘Race’ sample the GFP level was lower than in the + control indicating that the expression of GFP had indeed been inhibited (a 20% decrease for the ‘Race’ sample and an 11% decrease for the ‘MS2’ sample). The ‘GFP Dom’ sample however showed an approximate 8% increase in GFP fluorescence when compared to the + control. Although this is a bit unexpected is could be due to the fact that the GFP expression was initiated in the 1˚ set of conditions whereas it took place in the 2˚ for the + control. However it appears that no inhibition took place indicating that once GFP is being expressed the amount present of MS2 as expressed by Met17 is not able to significantly inhibit the level of GFP fluorescence.</p><br />
<h3.FACS Analysis</h3></div>Josephhttp://2010.igem.org/File:Fluorimeter_graph_for_Ms2_repression.jpgFile:Fluorimeter graph for Ms2 repression.jpg2010-10-08T18:55:32Z<p>Joseph: </p>
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<div></div>Josephhttp://2010.igem.org/MS2_coat-protein_effect_on_expression_of_GFP_in_pRS415MS2 coat-protein effect on expression of GFP in pRS4152010-10-08T18:53:24Z<p>Joseph: New page: {{:Team:Aberdeen_Scotland/css}} {{:Team:Aberdeen_Scotland/Title}} <html> <h1>Characterisation of the inhibition of GFP expression in pRS415 through the binding of the MS2 coat protein to ...</p>
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{{:Team:Aberdeen_Scotland/Title}}<br />
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<br />
<h1>Characterisation of the inhibition of GFP expression in pRS415 through the binding of the MS2 coat protein to MS2 stem loops</h1><br />
<h3>Aim</h3><br />
<p>The characterisation of the effect of MS2 on the expression of GFP by pRS415 will allow more accurate modelling of the system and will allow us to determine with more precision the probability of success of the cross-inhibition of the switch. Expressing MS2 using the Met17-MS2 vector will allow us to monitor the effect of MS2 without the complication of the λ-N-peptide produced by pRS415 in turn inhibiting the expression of MS2.</p><br />
<h3>Hypothesis</h3><br />
<p>The expression of MS2 by Met17-MS2 will result in a decrease in the level of expression of GFP by pRS415. The inhibition will show a linear correlation with the level of expression of MS2.<br />
<h3>Protocol</h3><br />
<p>During this experiment double transformants of BY4742 containing pRS415 and Met17-MS2 were used. Single transformants of BY4742, containing only pRS415, were used to provide the negative and positive controls for the expression of GFP.<br><br />
<br><br />
The double transformants were first cultured overnight in specific conditions in order to establish the desired pre-conditions. The cells were then washed and re-cultured in a different specific set of conditions which would allow the characterisation of the effect of MS2.<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/e/e5/Setup_for_MS2_charac.jpg"/><br />
</center><br />
<p>The different pre-established conditions allow us to determine whether the history of the sample affects the final result.<br><br />
<br><br />
Final samples were then washed and normalised before being analysed using microscopy, Fluospar Optima readings and FACS analysis.</p><br />
<h3>Results</h3><br />
<h3>Microscopy</h3><br />
<p>The microscopy analysis revealed that, in none of the samples, the GFP expression had been completely inhibited. All samples (bar the negative control) showed green fluorescence. The microscope did not allow us to determine if there was any variation however in the levels of GFP in each specific sample.</p> <br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/b1/GFP_fluo_under_MS2_repression.jpg"/><br />
</center></div>Josephhttp://2010.igem.org/File:GFP_fluo_under_MS2_repression.jpgFile:GFP fluo under MS2 repression.jpg2010-10-08T18:52:33Z<p>Joseph: </p>
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<div></div>Josephhttp://2010.igem.org/File:Setup_for_MS2_charac.jpgFile:Setup for MS2 charac.jpg2010-10-08T18:48:16Z<p>Joseph: </p>
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<div></div>Josephhttp://2010.igem.org/Team:Aberdeen_Scotland/ResultsTeam:Aberdeen Scotland/Results2010-10-08T18:43:24Z<p>Joseph: </p>
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<h1>Main Experimental Results</h1><br />
<p><br />
<b>Characterising the pRS414 related components of the switch</b><br><br />
</html><br />
[[Timed Induction of the Cup1 promoter using N4]]<br><br />
[[Copper Dose Response of the Cup1 promoter using N4]]<br><br />
<br />
<br><b>Characterising the pRS415 related components of the switch</b><br><br />
[[Galactose Dose Response of Gal1 promoter in pRS415]]<br><br />
[[Timed Induction of Gal1 promoter in pRS415]]<br><br />
<br><br />
<b>Characterising the mutual inhibition whithin the switch</b><br><br />
[[MS2 coat-protein effect on expression of GFP in pRS415]]<br><br />
<br><br />
<b>Troubleshooting pRS414</b><br><br />
[[Experimental Layout]]<br><br />
<br><br />
<b>Other Experiments</b><br><br />
[[Characterising Biobrick containing mOrange fluorescent protein]]</div>Josephhttp://2010.igem.org/Team:Aberdeen_Scotland/TeamMembersTeam:Aberdeen Scotland/TeamMembers2010-10-08T18:42:34Z<p>Joseph: </p>
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picture of Krystal<br />
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<h4>Krystal Annand</h4><br />
<p><b>BSc Chemistry: University of Aberdeen</b><br />
<br><br><br />
<p>Krystal has just completed her degree in Chemistry.</p><br />
</td><br />
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<img src="https://static.igem.org/mediawiki/2010/8/80/Brychan.jpg"/><br />
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<h4> Brychan Cromwell</h4><br />
<p><b>MEng Chemical Engineering (4th Year): University of Aberdeen</b><br />
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Brychan...</p><br />
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<img src="https://static.igem.org/mediawiki/2010/c/c4/Lisa_dryburgh.jpg"/><br />
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<h4> Lisa Dryburgh</h4><br />
<p><b>BSc Hons Maths and Physics (4th Year): University of Aberdeen</b><br />
<br><br><br />
Lisa has just completed her 3rd year.</p></td> <br />
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<img src="https://static.igem.org/mediawiki/2010/4/42/Me.jpg"/><br />
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<h4>Joseph Hoare</h4><br />
<p><b>BSc Hons Immunology (4th Year): University of Aberdeen</b><br />
<br><br><br />
Joseph...</p><br />
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<img src="https://static.igem.org/mediawiki/2010/2/27/Justyna.jpg"/><br />
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<h4>Justyna Kucia</h4><br />
<p><b>University of Aberdeen</b><br />
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Justyna ...</p><br />
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<img src="https://static.igem.org/mediawiki/2010/0/09/Stephen.jpg"/><br />
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<h4>Stephen Lam</h4><br />
<p><b>University of Aberdeen</b><br />
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Stephen ...</p><br />
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<img src="https://static.igem.org/mediawiki/2010/e/e7/Tina.jpg"/><br />
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<h4>Christina McLeman</h4><br />
<p><b>BSc Hons Geology, BSc Physics, Phd Physics and Geology (1st Year): University of Aberdeen</b><br />
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Tina ...</p><br />
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<img src="https://static.igem.org/mediawiki/2010/6/63/Ben_porter.jpg"/><br />
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<h4>Ben Porter</h4><br />
<p><b>BSc Hons Physics (4th Year): University of Aberdeen</b><br />
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Ben ...</p><br />
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<img src="https://static.igem.org/mediawiki/2010/1/17/Margaret-ann.jpg"/><br />
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<h4>Maragaret-Ann Seger</h4><br />
<p><b>BS Electrical Engineering (2nd Year): Olin College</b><br />
<br><br><br />
Margaret-Ann ...</p><br />
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<img src="https://static.igem.org/mediawiki/2010/0/06/Liz.jpg"/><br />
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<h4>Liz Threlkeld</h4><br />
<p><b>BS Maths and Biology (2nd Year): Olin College</b><br />
<br><br><br />
Liz ...</p><br />
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</html></div>Josephhttp://2010.igem.org/File:Me.jpgFile:Me.jpg2010-10-08T18:41:02Z<p>Joseph: </p>
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<div></div>Josephhttp://2010.igem.org/Copper_Dose_Response_of_the_Cup1_promoter_using_N4Copper Dose Response of the Cup1 promoter using N42010-10-08T18:27:33Z<p>Joseph: </p>
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<h1>Quantitative determination of the response of the CUP1 promoter to varying concentration of CuSO4</h1><br />
<br />
<h3>Aim</h3><br />
<p>The aim of this experiment is to characterise the CUP1 promoter present on N4 by determining whether<br />
it displays a dose response quality. The determined characteristics of this promoter can then be applied to the promoter present in pRS414 in order to allow more precise modelling of the switch.</p><br />
<br />
<h3>Hypothesis</h3><br />
<p>The CUP1 promoter ixhibits a linera relationship between the concentration of CuSO4 and the level of GFP expression when CuSO4 concentrations range from 0µM to 100µM. At concentrations of 100µM and hicher the expression level of GFP will have reached a steady state and will remain unchanged despite increasing CuSO4 concetrations. [1]</p><br />
<br />
<h3>Protocol</h3><br />
<p>A genomically integrated GFP gene under control of a CUP1 promoter was used to characterise the control properties of this promoter, this construct is referred to as N4 and was transformed into the yeast strain BY4741 for analysis.<br />
<br><br />
Three separate starter cultures were prepared of N4 in 5mL SD medium. the following tubes were then prepared in triplicate.</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/3/3a/Table1_for_Quantitative_determination_of_the_response_of_the_CUP1..._.png"/><br />
</center><br />
<p>Each one of these tubes was inoculated with N4 from each stater culture in order to provide triplicates of each concentratuion value. the cells were later harvested once the OD600 had reached 0.6. Triplicates from each culture were then loaded onto a 96 microtitre plate. The fluorescence of each sample was measured using a fluorometer running the "RussGFP" protocol.</p><br />
<h3>Results</h3><br />
<p>For full data spreadsheet<br><br />
IGEM 240610 JH+SL PCup Induction.xslx</p><br><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/2/20/Table2_for_Quantitative_determination_of_..._.png"/><br />
</center><br />
</p><br />
<h3>Conclusion</h3><br />
<p>The data suggests evidence of a dose dependent response. We can see from Figure 1that the relationship between the GFP levels and the copper concentrations is linear when the concentrations of CuSO4 range from 0µM to 75µM. At concentrations higher than 75µM the response seems to reach a plateau and the GFP levels no longer increase along with increasing copper concentrations. Figure 2 shows us that these characteristics appear for each individual culture.<br><br />
The data supports the hypothesis that the response of the copper promoter is dose dependent for a defined range of copper concentrations. The data however indicates that this range is smaller than our initial hypothesis suggested and that a plateau is reached with concentrations of 75μM as opposed to the initial 100μM.</p><br />
<h3>References</h3><br />
<p>[1]. Gorman JA, Clark PE, Lee MC, Debouck C and Rosenberg M<br><br />
Regulation of the yeast metallothionein gene<br><br />
Gene, 48 (19860 13-22<br><br />
<br><br />
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<b>[[https://2010.igem.org/Team:Aberdeen_Scotland/Results Return to Results Main page]]</b></div>Josephhttp://2010.igem.org/4._Replacing_the_CUP1_promoter_in_pRS414_with_the_CUP1-2_promoter_from_the_N4_construct4. Replacing the CUP1 promoter in pRS414 with the CUP1-2 promoter from the N4 construct2010-10-08T18:25:07Z<p>Joseph: </p>
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<h1>Checking the copper promoter in pRS414 by replacing it with the CUP1-2 promoter from the N4 construct</h1><br />
<h3>Aim</h3><br />
<p>When comparing the sequence of the promoter present in pRS414 to the sequence of the promoter in N4 we noticed that the N4 sequence contained 50 base pairs in its associated 5’UTR that were not present in the pRS414 5’UTR sequence. We have shown that the N4 promoter works (Characterisation of Cup1 Promoter experiments). By replacing the promoter in pRS414 with the promoter and associated 5’UTR from N4 we can determine whether or not pRS414 had a defective or incomplete promoter which resulted in no expression of CFP.</p><br />
<h3>Hypothesis</h3><br />
<p>The Cup promoter present in N4 contains fifty base pairs in its associated 5’UTR that are not present in the pRS414 construct which are responsible for the pRS414 construct not expressing CFP properly.</p><br />
<h3>Protocol</h3><br />
<p>The pRS414 construct was digested using the restriction enzymes Bgl2 and Pst1 in order to remove the existing Cup1 promoter. The promoter present in N4 (Cup1-2) and the associated 5’UTR were then PCR amplified using primers designed to add complementary overhangs to the gapped pRS414 construct to allow homologous recombination.</p> <br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/8/81/PRS414_construct.jpg"/><br />
</center><br />
<br><p>The gapped pRS414 vector and the PCR amplified Cup1-2 promoter were then co-transformed into yeast (BY4741ΔTrp strain) and incubated over several days. The resulting transformants were cultured in SD medium containing CuSO4 at concentrations high enough to reach full induction of the promoter. Final samples were washed and re-suspended in PBS and were then analysed using a microscope fitted with CFP filters.<br><br />
<h3>Results</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/0/0a/Absence_of_CFP_fluoresence.jpg"/><br />
</center><br />
<p>Microscope analysis revealed no fluorescence coming form the cell samples under the CFP excitation and filter settings. Controls containing pGAL/GFP showed clear fluorescence however only background fluorescence from the yeast cells was observed coming from the cells containing Cup1/CFP. 20 different samples were tested using different colonies form the transformation plate each time however all results came up negative. As we know that the promoter repaired into the pRS414 construct worked (see Characterisation of Cup1 Promoter experiments) we can conclude that the initial lack of CFP expression observed was not due to a faulty promoter but must stem from either the Bbox stem loop or the fusion of MS2 to CFP. <br />
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<b>[[https://2010.igem.org/Experimental_Layout Return to Troubleshooting pRS414 Main page]]</b><br><br />
<b>[[https://2010.igem.org/Team:Aberdeen_Scotland/Results Return to Results Main page]]</b></div>Josephhttp://2010.igem.org/File:Absence_of_CFP_fluoresence.jpgFile:Absence of CFP fluoresence.jpg2010-10-08T18:24:37Z<p>Joseph: </p>
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<div></div>Josephhttp://2010.igem.org/File:PRS414_construct.jpgFile:PRS414 construct.jpg2010-10-08T18:22:59Z<p>Joseph: </p>
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<div></div>Josephhttp://2010.igem.org/3._N-GFP_Swap_Experiment3. N-GFP Swap Experiment2010-10-08T18:13:56Z<p>Joseph: </p>
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{{:Team:Aberdeen_Scotland/Title}}<br />
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<h1>The N-GFP Swap Experiment</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment is to see if the pRS414 construct possesses a working promoter/5’leader/binding stem loop sequence by replacing the MS2-CFP part of the construct with the Npep-GFP sequence from pRS415. We have shown that Npep-GFP is expressed by pRS415 therefore if we can express it in pRS414 we can narrow down where the fault is located in pRS414.</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/7/77/Joint_diagram_of_pRS414%2BpRS415.jpg"/><br />
</center><br />
<h3>Hypothesis</h3><br />
<p>The reason why pRS414 is not expressing CFP lies within the promoter/5’leader/stem loop sequence.</p><br />
<h3>Protocol</h3><br />
<p>The pRS414 construct was digested using the restriction enzymes Cla1 and Nde1 in order to remove the MS2-CFP section of pRS414.<br><br />
In parallel, the Npeptide-GFP section of pRS415 was PCR amplified using primers designed to add 45 base pair long overhangs that would allow homologous recombination of the PCR product into the gapped pRS414. The gapped pRS414 and the PCR product were co-transformed into yeast (BY4741ΔTrp strain) in order to allow the homologous recombination to take place. The resulting transformants were then screened using Colony PCR in order to make sure that the swap had taken place.<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/b8/N-GFP_swap_construct.jpg"/><br />
</center><br />
<p>The colonies where the swap had been successful were then cultured overnight in SD medium containing Raffinose and CuSO4 (100µM). Samples were then normalised and resuspended in PBS and loaded onto a 96 well microtitre plate before being analysed using the FACS machine.</p><br />
<br />
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<h3>Results</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/c/cc/Fluor_graph_for_N-GFP_swap.jpg"/><br />
</center><br />
<p>From Fig 4. We can determine the level of background fluorescence of yeast cells using the results from the negative control “pRS415”, we can also see the expected levels of GFP in a working construct from the positive control of “pRS415 + 2% Gal”.<br><br />
The two samples from colony 10, which are in presence of Cu inducer, show readings that correspond with the levels of background fluorescence of yeast cells. This indicates that although the Swap was successful the pRS414 construct is still not expressing properly.<br> <br />
<br><br />
We can conclude from these conclude from these results that the problem resulting in pRS414 not being functional is related to either the Copper promoter or to the 5’UTR and Bbox stem loop. This experiment does not rule out however that the MS2-CFP part of the construct is also defective.<br />
<br />
<br><br />
<br />
</html><br />
<br><br />
<b>[[https://2010.igem.org/Experimental_Layout Return to Troubleshooting pRS414 Main page]]</b><br><br />
<b>[[https://2010.igem.org/Team:Aberdeen_Scotland/Results Return to Results Main page]]</b></div>Josephhttp://2010.igem.org/3._N-GFP_Swap_Experiment3. N-GFP Swap Experiment2010-10-08T18:12:49Z<p>Joseph: </p>
<hr />
<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<html><br />
<br />
<h1>The N-GFP Swap Experiment</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment is to see if the pRS414 construct possesses a working promoter/5’leader/binding stem loop sequence by replacing the MS2-CFP part of the construct with the Npep-GFP sequence from pRS415. We have shown that Npep-GFP is expressed by pRS415 therefore if we can express it in pRS414 we can narrow down where the fault is located in pRS414.</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/7/77/Joint_diagram_of_pRS414%2BpRS415.jpg"/><br />
</center><br />
<h3>Hypothesis</h3><br />
<p>The reason why pRS414 is not expressing CFP lies within the promoter/5’leader/stem loop sequence.</p><br />
<h3>Protocol</h3><br />
<p>The pRS414 construct was digested using the restriction enzymes Cla1 and Nde1 in order to remove the MS2-CFP section of pRS414.<br><br />
In parallel, the Npeptide-GFP section of pRS415 was PCR amplified using primers designed to add 45 base pair long overhangs that would allow homologous recombination of the PCR product into the gapped pRS414. The gapped pRS414 and the PCR product were co-transformed into yeast (BY4741ΔTrp strain) in order to allow the homologous recombination to take place. The resulting transformants were then screened using Colony PCR in order to make sure that the swap had taken place.<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/b8/N-GFP_swap_construct.jpg"/><br />
</center><br />
The colonies where the swap had been successful were then cultured overnight in SD medium containing Raffinose and CuSO4 (100µM). Samples were then normalised and resuspended in PBS and loaded onto a 96 well microtitre plate before being analysed using the FACS machine.<br />
<br />
<br><br />
<h3>Results</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/c/cc/Fluor_graph_for_N-GFP_swap.jpg"/><br />
</center><br />
<p>From Fig 4. We can determine the level of background fluorescence of yeast cells using the results from the negative control “pRS415”, we can also see the expected levels of GFP in a working construct from the positive control of “pRS415 + 2% Gal”.<br><br />
The two samples from colony 10, which are in presence of Cu inducer, show readings that correspond with the levels of background fluorescence of yeast cells. This indicates that although the Swap was successful the pRS414 construct is still not expressing properly.<br> <br />
<br><br />
We can conclude from these conclude from these results that the problem resulting in pRS414 not being functional is related to either the Copper promoter or to the 5’UTR and Bbox stem loop. This experiment does not rule out however that the MS2-CFP part of the construct is also defective.<br />
<br />
<br><br />
<br />
</html><br />
<br><br />
<b>[[https://2010.igem.org/Experimental_Layout Return to Troubleshooting pRS414 Main page]]</b><br><br />
<b>[[https://2010.igem.org/Team:Aberdeen_Scotland/Results Return to Results Main page]]</b></div>Josephhttp://2010.igem.org/File:Fluor_graph_for_N-GFP_swap.jpgFile:Fluor graph for N-GFP swap.jpg2010-10-08T18:12:09Z<p>Joseph: </p>
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<div></div>Josephhttp://2010.igem.org/File:N-GFP_swap_construct.jpgFile:N-GFP swap construct.jpg2010-10-08T18:10:56Z<p>Joseph: </p>
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<div></div>Joseph