Difference between revisions of "Part:BBa K2294007:Experience"

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==Characterization by BOKU-Vienna 2017==
 
==Characterization by BOKU-Vienna 2017==
  
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For characterization the synGAL promoter was combined with GFP and the CYC1 terminator and afterwards integrated into the <i>ura3 </i> gene of <i>Saccharomyces cerevisiae </i>. The GFP fluorescence was measured using a flow cytometer after inducing with 3 different galactose concentrations (0,5%, 1% and 2%).<br>
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For characterization the synGAL promoter was combined with <i>GFP</i> and the <i>CYC1</i> terminator and afterwards integrated into the <i>ura3 </i> gene of <i>Saccharomyces cerevisiae </i> to generate stable clones and avoid gene loss during GFP expression. The GFP fluorescence was measured using a flow cytometer after inducing with 3 different galactose concentrations (0.5%, 1% and 2%). In addition, the expression level of the pGAL was also investigated in YP + 2% glucose for determination of the backgroundlevel. Every condition (except of non induced and the wildtype) were cultivated in triplicates. <br>
 
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The cultivation was performded in 24 deepwell plates using 2 mL YP media. The preculture was performed in selective YPD. Prior to main culture inoculation the cells were washed twice in YP to remove remaining glucose. 100µL of 1mL preculture were used for inoculation of the main culture. As controls a wildtype and a clone expressing GFP under the constituve pTDH3 promoter were cultivated. In addition, the synGAL promoter without a Kozak sequence was investigated.
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The preculture was performed in 1 mL selective YPD. Prior to main culture inoculation the cells were washed twice in YP without any carbon source to remove remaining glucose. 100µL of the preculture were used for inoculation of 2 mL main culture which was cultivated at 30 °C at 280 rpm for around 5 hours. As controls a wildtype and a clone expressing GFP under the constituve pTDH3 promoter were cultivated. In addition, the synGAL promoter without a Kozak sequence was investigated. Before flow cytometer measurement cells were washed in PBS twice and afterwards diluted to a final OD of 0.5.  
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<br><br>
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Results: <br>
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<strong>Results:</strong> <br>
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[[File:BBa_K2294007_promoterstrength.png|900 px|thumb|center|<b>Figure 1:</b> Comparison of the GFP expression between pGAL with and without Kozak sequence under different galactose concentrasions and a comparison to the strenght of the pTDH3 at 2 % glucose.]]<br><br>
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[[File:BBa_K2294007_promoterstrength.png|900 px|thumb|center|<b>Figure 1:</b> Comparison of the GFP expressions between pGAL with and without Kozak sequence under different galactose concentrations and a comparison to the strenght of the pTDH3 at 2 % glucose.]]<br><br>
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There is no significant difference between the different galactose concentrations which were investigated. The galpromoter combined with a Kozak sequnce showed the highes expression levels of GFP. It is about twice compared to the gal promoter without a Kozak sequence and three times higher compared to the pTDH3 promoter of <i>Saccharomyces cerevisiae </i>. The clones expressing GFP under the control of the consitutive TDH3 promoter were cultivated in YP + 2% glucose.
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There is no significant difference between the different galactose concentrations which were investigated. The synGAL promoter combined with a Kozak sequnce showed the highest expression levels of GFP. It is about twice compared to the synGAL promoter without a Kozak sequence and three times higher compared to the pTDH3 promoter of <i>Saccharomyces cerevisiae </i>. The clones expressing GFP under the control of the consitutive TDH3 promoter were cultivated in YP + 2% glucose. The background expression level of both pGAL with and without Kozak sequence were very low.  
 
<br><br>[[File:BBa K2294007 comparison.png|1000px|thumb|center|<b>Figure 2:</b> left: Fowardscatter plotted against the fluorescence intensity. right: Histograms of forward scatter, side scatter and fluorescence.<br> <b>A:</b> fully induced pGAL  with Kozak sequence compared to the wildtype<br><b>B:</b> non induced pGAL with Kozak sequence compared to fully induced one<br> <b>C:</b> pGAL with Kozak sequnece (sample B3 36), pGAL without Kozak sequence (sample D4 37), pTDH3 and wildtype.    ]]
 
<br><br>[[File:BBa K2294007 comparison.png|1000px|thumb|center|<b>Figure 2:</b> left: Fowardscatter plotted against the fluorescence intensity. right: Histograms of forward scatter, side scatter and fluorescence.<br> <b>A:</b> fully induced pGAL  with Kozak sequence compared to the wildtype<br><b>B:</b> non induced pGAL with Kozak sequence compared to fully induced one<br> <b>C:</b> pGAL with Kozak sequnece (sample B3 36), pGAL without Kozak sequence (sample D4 37), pTDH3 and wildtype.    ]]
 
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Latest revision as of 10:46, 1 November 2017


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Characterization by BOKU-Vienna 2017

For characterization the synGAL promoter was combined with GFP and the CYC1 terminator and afterwards integrated into the ura3 gene of Saccharomyces cerevisiae to generate stable clones and avoid gene loss during GFP expression. The GFP fluorescence was measured using a flow cytometer after inducing with 3 different galactose concentrations (0.5%, 1% and 2%). In addition, the expression level of the pGAL was also investigated in YP + 2% glucose for determination of the backgroundlevel. Every condition (except of non induced and the wildtype) were cultivated in triplicates.

The preculture was performed in 1 mL selective YPD. Prior to main culture inoculation the cells were washed twice in YP without any carbon source to remove remaining glucose. 100µL of the preculture were used for inoculation of 2 mL main culture which was cultivated at 30 °C at 280 rpm for around 5 hours. As controls a wildtype and a clone expressing GFP under the constituve pTDH3 promoter were cultivated. In addition, the synGAL promoter without a Kozak sequence was investigated. Before flow cytometer measurement cells were washed in PBS twice and afterwards diluted to a final OD of 0.5.

Results:

Figure 1: Comparison of the GFP expressions between pGAL with and without Kozak sequence under different galactose concentrations and a comparison to the strenght of the pTDH3 at 2 % glucose.


There is no significant difference between the different galactose concentrations which were investigated. The synGAL promoter combined with a Kozak sequnce showed the highest expression levels of GFP. It is about twice compared to the synGAL promoter without a Kozak sequence and three times higher compared to the pTDH3 promoter of Saccharomyces cerevisiae . The clones expressing GFP under the control of the consitutive TDH3 promoter were cultivated in YP + 2% glucose. The background expression level of both pGAL with and without Kozak sequence were very low.



Figure 2: left: Fowardscatter plotted against the fluorescence intensity. right: Histograms of forward scatter, side scatter and fluorescence.
A: fully induced pGAL with Kozak sequence compared to the wildtype
B: non induced pGAL with Kozak sequence compared to fully induced one
C: pGAL with Kozak sequnece (sample B3 36), pGAL without Kozak sequence (sample D4 37), pTDH3 and wildtype.