Difference between revisions of "Part:BBa K122000"
 
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INTRODUCTION
 
INTRODUCTION
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<p>In order to determine the strength of pPGK1 promoter, we have conducted the following experiment. First of all, we have constructed 2 plasmids where EGFP is expressed under different constitutive promoters. As a positive control, we used pTDH3. The empty plasmid was used as a negative control.
+
<p>pPGK1 is a constitutive promoter. In order to determine the strength of pPGK1 promoter, we have conducted the following experiment. First of all, we have constructed a plasmid where EGFP is expressed under the pPGK1 promoter. The empty plasmid was used as a negative control.
  
−
</p><p>MATERIALS AND METHODS
+
</p><p>Methodology
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</p><p>Plasmids used to conduct the experiment are listed in table 1.
+
  
−
Table 1. Plasmids created
+
All common methods such as PCR, agarose gel electrophoresis, miniprep, bacterial and yeast transformation, and some others are described in the Methodology section in Tartu_TUIT2019 wiki page.
−
{| class="wikitable"
+
 
−
|colspan="2"|
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Plasmid construction
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|colspan="3"|Insert
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Target promoters were PCR amplified from yeast genomic DNA with primers containing SacI (forward primer) and NotI (reverse primer) restriction sites at their 5’-ends. PCR products were separated on the agarose gel, purified and cut with SacI/NotI enzymes. As a backbone, we used pRS306 plasmid containing pTDH3-GFP-tCYC1 cassette. The vector was digested with SacI/NotI restriction enzymes, which cut out the pTDH3 promoter region, and was ligated with our target promoter. After bacterial transformation and miniprep, sequence-verified plasmids were used for yeast transformation.
−
|
+
−
|-
+
−
|number
+
−
|Plasmid name
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−
|Promoter
+
−
|Gene
+
−
|Terminator
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−
|backbone
+
−
|-
+
−
|1
+
−
|pRS306 pPGK1-EGFP-tCYC1
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−
|pPGK1
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−
|EGFP
+
−
|tCYC1
+
−
|pRS306
+
−
|-
+
−
|2
+
−
|pRS306
+
−
|pPGK1
+
−
|-
+
−
|-
+
−
|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).
+
Yeast strain construction
 +
 
 +
Constructed vectors with GFP under target promoters were restricted and used for yeast transformation. <i>S. cerevisiae</i> DOM90 <i>(MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+])</i> strain was transformed. All the yeast strains generated and used for promoter characterization are listed in Table 1.  
 +
 
 +
Table 1. Yeast strains used for promoter characterization experiments
  
−
Table 2. ''S. cerevisiae'' strains created.
 
 
{| class="wikitable"
 
{| class="wikitable"
−
|
+
|Strain name
−
|Strain name
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|Genotype*
−
|Genotype
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|Description
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|Plasmid integrated
+
 
|-
 
|-
−
|Negative control
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|DOM0090
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|DOM90
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|<i>ura3</i>-1::pRS306-URA3
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|MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]
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|Strain with empty vector; was used as a negative control for GFP fluorescence
−
|pRS306
+
 
|-
 
|-
−
|Positive control(pTDH3)
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|TTI_1
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|DOM90
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|<i>ura3</i>-1::pRS306-pTDH3-EGFP-tCYC1-URA3
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|MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]
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|Strain with EGFP under TDH3 promoter; was used as a positive control for GFP fluorescence
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|pRS306
+
 
|-
 
|-
−
|pPGK1
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|TTI_4
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|DOM90
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|<i>ura3</i>-1::pRS306 pPGK1-EGFP-tCYC1-URA3
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|MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]
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|Strain with GFP under pPGK1 promoter
−
|pRS306
+
 
|}
 
|}
  
−
We have measured the EGFP fluorescence intensity with BioTek Synergy MX microplate reader.
+
*DOM90 strain <i>(MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+])</i> was used for transformation. In the Genotype column, only the differences between strains are provided.
 +
Following transformation, separate yeast colonies were screened for the presence of the insert under the fluorescent microscope. Colonies displaying GFP fluorescence were selected for further fluorescence measurement experiment.
  
−
All strains were pregrown overnight at 30°C in liquid CSM/2%Glc media, resuspended in fresh CSM/2%Glc media to OD600 in the range of 0.8 to 1. 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.
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Fluorescence measurement
  
−
Table 3. The 96 well plate layout for OD600 and Fluorescence measurements.
+
Prior to measurements, yeast strains were pregrown in liquid media overnight at 30°C and 200 RPM in the shaker incubator. The next day, optical density (OD600) of yeast cultures was measured. Strains were diluted with fresh media to reach the final OD600 from 0.8 to 1.1. Before the fluorescence measurements, cell cultures were grown for 3 hours at 30°C and 200 RPM in the shaker incubator.  
 +
 
 +
All the media used for different strains are listed in Table 2.
 +
 +
Table 2. Media used for cell growth
 
{| class="wikitable"
 
{| class="wikitable"
−
|Negative control
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|Strain
−
|Positive control
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|Medium
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|pPGK1
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|Carbon source
−
|
+
 
|-
 
|-
−
|
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|<i>ura3</i>-1::pRS306-URA3
−
|
+
|CSM
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|Colony 1 Replicate 1
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|2% glucose
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|Colony 2 Replicate 1
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|-
 
|-
−
|
+
|<i>ura3</i>-1::pRS306-pTDH3-EGFP-tCYC1-URA3
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|
+
|CSM
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|Colony 1 Replicate 2
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|2% glucose
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|Colony 2 Replicate 2
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|-
 
|-
−
|
+
|<i>ura3</i>-1::pRS306 pPGK1-EGFP-tCYC1-URA3
−
|
+
|CSM
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|Colony 1 Replicate 3
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|2% glucose
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|Colony 2 Replicate 3
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−
|-
+
−
|
+
−
|
+
−
|Colony 1 Replicate 4
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−
|Colony 2 Replicate 4
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−
|-
+
−
|
+
−
|
+
−
|Colony 1 Replicate 5
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−
|Colony 2 Replicate 5
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−
|-
+
−
|
+
−
|
+
−
|Colony 1 Replicate 6
+
−
|Colony 2 Replicate 6
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−
|-
+
−
|
+
−
|
+
−
|Colony 1 Replicate 7
+
−
|Colony 2 Replicate 7
+
−
|-
+
−
|
+
−
|
+
−
|Colony 1 Replicate 8
+
−
|Colony 2 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.  
+
After 3 hours of incubation, 200 µL of each strain culture was transferred into a 96-well plate (clear flat bottom). Two biological replicates (two different positive colonies after transformation) of each genotype were taken in four technical replicates for fluorescence measurements. 
−
 
+
For the fluorescence measurement, we have used BioTek Synergy MX microplate reader with the following setup: excitation 485 nm, emission 528 nm, bandwidth 20, gain 80. Alone with fluorescence, absorbance at 600 nm was measured (with disabled pathlength correction). CSM medium was used as a blank for OD600 measurements. Fluorescein was used as a reference (according to the protocol of 2018 iGEM InterLab Study) to quantify GFP fluorescence intensity. However, in our case, the standard curve for fluorescein concentrations in the range from 0.0012 to 0.313 µM was generated, in contrast to the InterLab Study, where 10 µM of fluorescein was the highest concentration. Since we used a higher gain parameter in our experiment, fluorescein concentrations above 0.313 µM showed intensities that were above the detection limit of our device.  
−
 
+
  
−
 
+
Results
−
    [File:pPGK1.jpg|500px]
+
  
 +
EGFP was cloned under target promoter and constructs were transformed into yeast cells. The intensity of GFP fluorescence was taken as a measure of promoter strength.
 +
As can be seen from Figure 1, EGFP under pPGK1 showed 72% relative to EGFP fluorescence in the positive control (pTDH3-EGFP).
  
−
CONCLUSIONS
+
Figure 1.
−
<p> The results are as expected, negative control does not show almost any fluorescence. The promoter shows fluorescence a bit less than the positive control.
+
[[File:pPGK1.jpg|500px]]

Latest revision as of 11:32, 19 October 2019


pPGK1

This is the 1500 bp upstream of the yeast PGK1 coding region. This is a putative constitutive promoter.

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]


INTRODUCTION

pPGK1 is a constitutive promoter. In order to determine the strength of pPGK1 promoter, we have conducted the following experiment. First of all, we have constructed a plasmid where EGFP is expressed under the pPGK1 promoter. The empty plasmid was used as a negative control.

Methodology

All common methods such as PCR, agarose gel electrophoresis, miniprep, bacterial and yeast transformation, and some others are described in the Methodology section in Tartu_TUIT2019 wiki page.

Plasmid construction Target promoters were PCR amplified from yeast genomic DNA with primers containing SacI (forward primer) and NotI (reverse primer) restriction sites at their 5’-ends. PCR products were separated on the agarose gel, purified and cut with SacI/NotI enzymes. As a backbone, we used pRS306 plasmid containing pTDH3-GFP-tCYC1 cassette. The vector was digested with SacI/NotI restriction enzymes, which cut out the pTDH3 promoter region, and was ligated with our target promoter. After bacterial transformation and miniprep, sequence-verified plasmids were used for yeast transformation.

Yeast strain construction

Constructed vectors with GFP under target promoters were restricted and used for yeast transformation. S. cerevisiae DOM90 (MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]) strain was transformed. All the yeast strains generated and used for promoter characterization are listed in Table 1.

Table 1. Yeast strains used for promoter characterization experiments

Strain name Genotype* Description
DOM0090 ura3-1::pRS306-URA3 Strain with empty vector; was used as a negative control for GFP fluorescence
TTI_1 ura3-1::pRS306-pTDH3-EGFP-tCYC1-URA3 Strain with EGFP under TDH3 promoter; was used as a positive control for GFP fluorescence
TTI_4 ura3-1::pRS306 pPGK1-EGFP-tCYC1-URA3 Strain with GFP under pPGK1 promoter
  • DOM90 strain (MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]) was used for transformation. In the Genotype column, only the differences between strains are provided.

Following transformation, separate yeast colonies were screened for the presence of the insert under the fluorescent microscope. Colonies displaying GFP fluorescence were selected for further fluorescence measurement experiment.

Fluorescence measurement

Prior to measurements, yeast strains were pregrown in liquid media overnight at 30°C and 200 RPM in the shaker incubator. The next day, optical density (OD600) of yeast cultures was measured. Strains were diluted with fresh media to reach the final OD600 from 0.8 to 1.1. Before the fluorescence measurements, cell cultures were grown for 3 hours at 30°C and 200 RPM in the shaker incubator.

All the media used for different strains are listed in Table 2.

Table 2. Media used for cell growth

Strain Medium Carbon source
ura3-1::pRS306-URA3 CSM 2% glucose
ura3-1::pRS306-pTDH3-EGFP-tCYC1-URA3 CSM 2% glucose
ura3-1::pRS306 pPGK1-EGFP-tCYC1-URA3 CSM 2% glucose

After 3 hours of incubation, 200 µL of each strain culture was transferred into a 96-well plate (clear flat bottom). Two biological replicates (two different positive colonies after transformation) of each genotype were taken in four technical replicates for fluorescence measurements. For the fluorescence measurement, we have used BioTek Synergy MX microplate reader with the following setup: excitation 485 nm, emission 528 nm, bandwidth 20, gain 80. Alone with fluorescence, absorbance at 600 nm was measured (with disabled pathlength correction). CSM medium was used as a blank for OD600 measurements. Fluorescein was used as a reference (according to the protocol of 2018 iGEM InterLab Study) to quantify GFP fluorescence intensity. However, in our case, the standard curve for fluorescein concentrations in the range from 0.0012 to 0.313 µM was generated, in contrast to the InterLab Study, where 10 µM of fluorescein was the highest concentration. Since we used a higher gain parameter in our experiment, fluorescein concentrations above 0.313 µM showed intensities that were above the detection limit of our device.

Results

EGFP was cloned under target promoter and constructs were transformed into yeast cells. The intensity of GFP fluorescence was taken as a measure of promoter strength. As can be seen from Figure 1, EGFP under pPGK1 showed 72% relative to EGFP fluorescence in the positive control (pTDH3-EGFP).

Figure 1.

PPGK1.jpg