Difference between revisions of "Part:BBa K165000"
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ITRODUCTION
 
ITRODUCTION
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<p>In order to determine the strength of pMET25 promoter, we have conducted the following experiment. First of all, we have constructed 2 plasmids where EGFP is expressed under different constitutive promoters. We have chosen pTDH3 as a positive control due to its wide use in yeast research. The empty plasmid was used as a negative control.
+
<p>In order to determine the strength of pMET25 promoter, we have conducted the following experiment. First of all, we have constructed 2 plasmids where EGFP is expressed under different constitutive promoters. The empty plasmid was used as a negative control.
  
−
</p><p>MATERIAL AND METHOD
+
</p><p>MATERIALS AND METHODS
 
</p><p>Plasmids used to conduct the experiment are listed in table 1.
 
</p><p>Plasmids used to conduct the experiment are listed in table 1.
  
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|-
 
|-
 
|1
 
|1
−
|pRS306 pTDH3-EGFP-tCYC1
+
|pRS306 pMET25-EGFP-tCYC1
−
|TDH3
+
|MET25
 
|EGFP
 
|EGFP
 
|tCYC1
 
|tCYC1
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|-
 
|-
 
|2
 
|2
−
|pRS306 pRPL18B-EGFP-tCYC1
+
|pRS306
−
|MET25
+
|MET25
−
|EGFP
+
|-
−
|tCYC1
+
|-
 
|pRS306
 
|pRS306
 
|}
 
|}
  
−
After construction of plasmids was finished, we have transformed ''S. cerevisiae'' CEN.PK-2-1C strain with plasmids listed above to create the strains (table 2).
+
After construction of plasmids was finished, we have transformed ''S. cerevisiae'' DOM90 (MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]) strain with plasmids listed above to create the strains (table 2).
  
 
Table 2. ''S. cerevisiae'' strains created.
 
Table 2. ''S. cerevisiae'' strains created.
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|Plasmid integrated
 
|Plasmid integrated
 
|-
 
|-
−
|Positive control
+
|Negative control
−
|
+
|DOM90
−
|
+
|MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]
−
|
+
|pRS306
 
|-
 
|-
−
|Negative control
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|Met25 uninduced
−
|
+
|DOM90
−
|
+
|MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]
 
|pRS306
 
|pRS306
 
|-
 
|-
−
|Test
+
|Met25 induced
−
|
+
|DOM90
−
|
+
|MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]
−
|
+
|pRS306
 
|}
 
|}
  
 
We have measured the EGFP fluorescence intensity with BioTek Synergy MX microplate reader.
 
We have measured the EGFP fluorescence intensity with BioTek Synergy MX microplate reader.
  
−
All strains were pregrown overnight at 30°C in liquid CSM/2%Glc media, resuspended in fresh CSM/2%Glc media to OD600 ca. 0.7, divided into 2 parts: half was induced by changing media from  CSM to -Met, half was left uninduced. Distributed to 96 well plate (clear flat bottom). 8 replicates from the strain were used. 200 µL of cells were added to each well. As a reference to OD600, fresh CSM/2%Glc media was used. After the 96 well plate was ready, the OD600 and fluorescence (excitation 485 nm, emission 528 nm, bandwidth 20, gain 80) were measured. The layout of the plate is described in table 3.
+
All strains were pregrown overnight at 30°C in liquid CSM/2%Glc media, resuspended in fresh CSM/2%Glc media to OD600 ca. 0.7, divided into 2 parts: half was induced by changing media from  CSM to -Met, half was left uninduced. Put to grow for 3 hours. Distributed to 96 well plate (clear flat bottom). 8 replicates from the strain were used. 200 µL of cells were added to each well. As a reference to OD600, fresh CSM/2%Glc media was used. After the 96 well plate was ready, the OD600 and fluorescence (excitation 485 nm, emission 528 nm, bandwidth 20, gain 80) were measured. The layout of the plate is described in table 3.
  
 
Table 3. The 96 well plate layout for OD600 and Fluorescence measurements.
 
Table 3. The 96 well plate layout for OD600 and Fluorescence measurements.
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|
 
|
 
|Negative control
 
|Negative control
−
|Positive control(pTDH3)
+
|Met25 uninduced
−
|MET25
+
|MET25 induced
 
|-
 
|-
 
|Colony 1 Replicate 1
 
|Colony 1 Replicate 1
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As a fluorescence reference, the standard curve of fluorescence for fluorescein concentration was generated according to 2018 iGEM InterLab Study. The only difference from the InterLab protocol was the concentration of the fluorescein used. In the InterLab study, the highest fluorescein concentration used was 10 µM. But due to the higher gain parameter used in our measurement experiment, we have used 0.313 µM to avoid out of range measurements.  
 
As a fluorescence reference, the standard curve of fluorescence for fluorescein concentration was generated according to 2018 iGEM InterLab Study. The only difference from the InterLab protocol was the concentration of the fluorescein used. In the InterLab study, the highest fluorescein concentration used was 10 µM. But due to the higher gain parameter used in our measurement experiment, we have used 0.313 µM to avoid out of range measurements.  
  
−
<h1>Image Demo</h1>
+
 
−
   <p>
+
 
−
     <img src = "MET25.jpg"
+
    
−
        alt = "Picture of a happy monkey" />
+
     [[File:Met25.jpg|500px]]
 +
 
 +
 
 +
CONCLUSIONS
 +
<p> The results are as expected, negative control does not show almost any fluorescence. Uninduced pMET25 shows basic flourescence which can be due to the leaking of promoter. However induced promoter shows high fluorescence.

Revision as of 07:27, 9 October 2019

MET 25 Promoter 


 The is a repressable promoter for use in Saccharomyces cerevisiae.  It is repressed by the presence of methionine.  When unrepresssed, is has a moderate level of activity.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


ITRODUCTION

In order to determine the strength of pMET25 promoter, we have conducted the following experiment. First of all, we have constructed 2 plasmids where EGFP is expressed under different constitutive promoters. The empty plasmid was used as a negative control.

MATERIALS AND METHODS

Plasmids used to conduct the experiment are listed in table 1.

Table 1. Plasmids created

Insert
number Plasmid name Promoter Gene Terminator backbone
1 pRS306 pMET25-EGFP-tCYC1 MET25 EGFP tCYC1 pRS306
2 pRS306 MET25
pRS306

After construction of plasmids was finished, we have transformed S. cerevisiae DOM90 (MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]) strain with plasmids listed above to create the strains (table 2).

Table 2. S. cerevisiae strains created.

Strain name Genotype Plasmid integrated
Negative control DOM90 MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+] pRS306
Met25 uninduced DOM90 MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+] pRS306
Met25 induced DOM90 MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+] pRS306

We have measured the EGFP fluorescence intensity with BioTek Synergy MX microplate reader.

All strains were pregrown overnight at 30°C in liquid CSM/2%Glc media, resuspended in fresh CSM/2%Glc media to OD600 ca. 0.7, divided into 2 parts: half was induced by changing media from CSM to -Met, half was left uninduced. Put to grow for 3 hours. Distributed to 96 well plate (clear flat bottom). 8 replicates from the strain were used. 200 µL of cells were added to each well. As a reference to OD600, fresh CSM/2%Glc media was used. After the 96 well plate was ready, the OD600 and fluorescence (excitation 485 nm, emission 528 nm, bandwidth 20, gain 80) were measured. The layout of the plate is described in table 3.

Table 3. The 96 well plate layout for OD600 and Fluorescence measurements.

Negative control Met25 uninduced MET25 induced
Colony 1 Replicate 1
Colony 1 Replicate 2
Colony 1 Replicate 3
Colony 1 Replicate 4
Colony 1 Replicate 5
Colony 1 Replicate 6
Colony 1 Replicate 7
Colony 1 Replicate 8

As a fluorescence reference, the standard curve of fluorescence for fluorescein concentration was generated according to 2018 iGEM InterLab Study. The only difference from the InterLab protocol was the concentration of the fluorescein used. In the InterLab study, the highest fluorescein concentration used was 10 µM. But due to the higher gain parameter used in our measurement experiment, we have used 0.313 µM to avoid out of range measurements. 



   Met25.jpg


CONCLUSIONS

<p> The results are as expected, negative control does not show almost any fluorescence. Uninduced pMET25 shows basic flourescence which can be due to the leaking of promoter. However induced promoter shows high fluorescence.