MS2 Coat-Protein Effect on Expression of GFP in pRS415

From 2010.igem.org

(Difference between revisions)
Line 20: Line 20:
'''<u>Microscopy</u>'''<br>
'''<u>Microscopy</u>'''<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>
 +
<center>
 +
http://2010.igem.org/wiki/images/c/c0/MS2vsGFP_fluorescent_cells.jpg
 +
</center>
 +
'''<u>Fluospar Optima Readings</u>'''<br>
 +
The fluorimeter readings correlated the microscopy results by recording fluorescence in all samples except the – control.
 +
<center>
 +
http://2010.igem.org/wiki/images/5/5a/MS2vsGFP_fluorimeter.jpg
 +
</center>
 +
<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>

Revision as of 12:57, 24 October 2010

University of Aberdeen - ayeSwitch - iGEM 2010

Contents

Characterising the translational repression of GAL1p-[Npeptide-GFP] by trans expression of the MS2 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 was 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.