MS2 Coat-Protein Effect on Expression of GFP in pRS415

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<h1> Characterising the translational repression of GAL1p-[Npeptide-GFP] by trans expression of the MS2 protein </h1>
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<h1> Characterising the Translational Repression of GAL1p-[Npeptide-GFP] by Trans Expression of the MS2 Coat Protein </h1>
<h3>Aim</h3>
<h3>Aim</h3>
<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>
<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>
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<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>
<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>
<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>
<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>
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http://2010.igem.org/wiki/images/0/04/Conditions_for_FACS_analysis_of_MS2vsGFP.jpg
http://2010.igem.org/wiki/images/0/04/Conditions_for_FACS_analysis_of_MS2vsGFP.jpg
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<p>* 500μM Met was used as this concentration was been used in other experiments to successfully completely switch of the Met17 promoter [1]. <br>
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<p>* 500μM Met was used as this concentration has been used in other experiments to successfully completely switch of the Met17 promoter<a href="#ref1"><sup style="font-size:10px">[1]</sup></a>. <br>
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The different pre-established conditions allow us to determine whether the history of the sample affects the final result.<br>
The different pre-established conditions allow us to determine whether the history of the sample affects the final result.<br>
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<h3>Results</h3>
<h3>Results</h3>
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'''<u>Microscopy</u>'''<br>
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<h5><u>Microscopy</u><h5><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>
<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>
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http://2010.igem.org/wiki/images/c/c0/MS2vsGFP_fluorescent_cells.jpg
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'''<u>Fluospar Optima Readings</u>'''<br>
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The fluorimeter readings correlated the microscopy results by recording fluorescence in all samples except the – control.
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<h5><u>Fluospar Optima Readings</u></h5><br>
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The fluorimeter readings correlated the microscopy results by recording fluorescence in all samples except the – control.<br>
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http://2010.igem.org/wiki/images/5/5a/MS2vsGFP_fluorimeter.jpg
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<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>
<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>
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<br>
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<h5><u>FACS analysis</u></h5>
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<br>
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<p>The first <a href="http://2010.igem.org/FACS_analysis_of_fluorescent_proteins"><i>FACS</i></a></> 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>
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<p>The second <a href="http://2010.igem.org/FACS_analysis_of_fluorescent_proteins"><i>FACS</i></a></> 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>
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http://2010.igem.org/wiki/images/d/d8/MS2vsGFP_FACS_2.jpg
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<br><br>
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<p>The results showed that the inhibition of GFP expression by GAL1p-[Npeptide-GFP] by MS2 previously seen (see Fig.3) is indeed dependent on the concentration of MS2.<br>
 +
<br>
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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>
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<h3>References</h3>
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<a name="ref1"></a>
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<p>[1] Dominik Mumberg, Rolf MulIer and Martin Funk*<br>
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Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression<br>
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Nucleic Acids Research, 1994, Vol. 22, No. 25 5767-5768</p>
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<a href="http://2010.igem.org/Team:Aberdeen_Scotland/Results"><img src="http://2010.igem.org/wiki/images/8/8e/Left_arrow.png">&nbsp;&nbsp;Return to Results Main Page</a>
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{{:Team:Aberdeen_Scotland/Footer}}

Latest revision as of 19:55, 27 October 2010

University of Aberdeen - ayeSwitch - iGEM 2010

Characterising the Translational Repression of GAL1p-[Npeptide-GFP] by Trans Expression of the MS2 Coat Protein

Aim

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.

Hypothesis

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.

Protocol

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.


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.


Conditions_for_FACS_analysis_of_MS2vsGFP.jpg

* 500μM Met was used as this concentration has been used in other experiments to successfully completely switch of the Met17 promoter[1].

The different pre-established conditions allow us to determine whether the history of the sample affects the final result.
Final samples were then washed and normalised before being analysed using microscopy, Fluospar Optima readings and FACS analysis.


Results

Microscopy

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.

MS2vsGFP_fluorescent_cells.jpg

Fluospar Optima Readings

The fluorimeter readings correlated the microscopy results by recording fluorescence in all samples except the – control.

MS2vsGFP_fluorimeter.jpg


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.


FACS analysis

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.


MS2vsGFP_FACS_1.jpg


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.


MS2vsGFP_FACS_2.jpg



The results showed that the inhibition of GFP expression by GAL1p-[Npeptide-GFP] by MS2 previously seen (see Fig.3) is indeed dependent on the concentration of MS2.

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).

References

[1] Dominik Mumberg, Rolf MulIer and Martin Funk*
Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression
Nucleic Acids Research, 1994, Vol. 22, No. 25 5767-5768





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