Difference between revisions of "Part:BBa K165000"

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−	The is a repressible promoter for use in Saccharomyces cerevisiae.  It is repressed by the presence of methionine.  When unrepressed, is has a moderate level of activity.	+	The is a repressible promoter for use in <i>Saccharomyces cerevisiae</i>.  It is repressed by the presence of methionine.  When unrepressed, is has a moderate level of activity.
	<!-- Add more about the biology of this part here		<!-- Add more about the biology of this part here
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	INTRODUCTION		INTRODUCTION
−	<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>pMET25 is inducible(silent when methionine is present in the growth medium and is activated in methionine drop-out medium). In order to determine the strength of pMET25 promoter, we have conducted the following experiment. First of all, we have constructed a plasmid where EGFP is expressed under MET25 promoter. The empty plasmid was used as a negative control.
−	</p><p>MATERIALS AND METHODS	+	</p><p>Methodology
−	</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"|	+	Plasmid construction
−	|colspan="3"|Insert	+	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
−	|number	+
−	|Plasmid name	+
−	|Promoter	+
−	|Gene	+
−	|Terminator	+
−	|backbone	+
−	|-	+
−	|1	+
−	|pRS306 pMET25-EGFP-tCYC1	+
−	|MET25	+
−	|EGFP	+
−	|tCYC1	+
−	|pRS306	+
−	|-	+
−	|2	+
−	|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).	+	Constructed vectors with GFP under target promoters were restricted and used for yeast transformation.<i>S. cerevisiae</i> DOM90 (MATa <i>{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	+	|Genotype*
−	|Genotype	+	|Description
−	|Plasmid integrated	+
	|-		|-
−	|Negative control	+	|DOM0090
−	|DOM90	+	|<i>ura3</i>-1::pRS306-URA3
−	|MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]	+	|Strain with empty vector; was used as a negative control for GFP fluorescence
−	|pRS306	+
	|-		|-
−	|Met25 uninduced	+	|TTI_1
−	|DOM90	+	|<i>ura3</i>-1::pRS306-pTDH3-EGFP-tCYC1-URA3
−	|MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]	+	|Strain with EGFP under TDH3 promoter; was used as a positive control for GFP fluorescence
−	|pRS306 pMET25-EGFP-tCYC1	+
	|-		|-
−	|Met25 induced	+	|TTI_2
−	|DOM90	+	|<i>ura3</i>-1::pRS306 pMET25-EGFP-tCYC1-URA3
−	|MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]	+	|Strain with GFP under pMET25 promoter
−	|pRS306 pMET25-EGFP-tCYC1	+
	|}		|}
−	We have measured the EGFP fluorescence intensity with BioTek Synergy MX microplate reader.	+	*DOM90 strain (MATa <i>{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 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.	+	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. Proper culture volumes were first centrifuged, washed with distilled water and resuspended in either inductive or non-inductive media to get final culture 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"
−	|	+	|Strain
−	|Negative control	+	|Medium
−	|Met25 uninduced	+	|Carbon source
−	|MET25 induced	+
−	|SCM	+
	|-		|-
−	|Colony 1 Replicate 1	+	|<i>ura3</i>-1::pRS306-URA3
−	|	+	|CSM
−	|	+	|2% glucose
−	|	+
−	|	+
	|-		|-
−	|Colony 1 Replicate 2	+	|<i>ura3</i>-1::pRS306-pTDH3-EGFP-tCYC1-URA3
−	|	+	|CSM
−	|	+	|2% glucose
−	|	+
−	|	+
	|-		|-
−	|Colony 1 Replicate 3	+	|<i>ura3</i>-1::pRS306 pMET25-EGFP-tCYC1-URA3
−	|	+	|CSM (non-inductive conditions)
−	|	+	CSM methionine drop-out(inductive conditions)
−	|	+	|2% glucose
−	|	+
−	|-	+
−	|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.	+	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 in figure 1, EGFP under induced pMet25 showed 40% higher fluorescence in comparison to a positive control (pTDH3-EGFP). Surprisingly, EGFP fluorescence was detected in the strains with the inducible promoter under non-inductive conditions (presence of methionine).
−		+	Figure 1.
−	[[File:Met25.jpg|500px]]	+	[[File:uninMet25.jpg|500px]]
	CONCLUSIONS		CONCLUSIONS
−	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.	+
		+	The results of EGFP fluorescence measurements suggest that characterized yeast promoter initiate transcription at the rate, which was higher in our experiment than one observed for pTDH3 (positive control). Surprisingly, pMET25 promoter showed some leakage effect under non-inductive conditions that should be taken into consideration while choosing a promoter for the experiment.

---

Latest revision as of 11:35, 19 October 2019

MET 25 Promoter

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

Sequence and Features

INTRODUCTION

pMET25 is inducible(silent when methionine is present in the growth medium and is activated in methionine drop-out medium). In order to determine the strength of pMET25 promoter, we have conducted the following experiment. First of all, we have constructed a plasmid where EGFP is expressed under MET25 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_2	ura3-1::pRS306 pMET25-EGFP-tCYC1-URA3	Strain with GFP under pMET25 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. Proper culture volumes were first centrifuged, washed with distilled water and resuspended in either inductive or non-inductive media to get final culture 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 pMET25-EGFP-tCYC1-URA3	CSM (non-inductive conditions) CSM methionine drop-out(inductive conditions)	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 in figure 1, EGFP under induced pMet25 showed 40% higher fluorescence in comparison to a positive control (pTDH3-EGFP). Surprisingly, EGFP fluorescence was detected in the strains with the inducible promoter under non-inductive conditions (presence of methionine).

Figure 1.

CONCLUSIONS

The results of EGFP fluorescence measurements suggest that characterized yeast promoter initiate transcription at the rate, which was higher in our experiment than one observed for pTDH3 (positive control). Surprisingly, pMET25 promoter showed some leakage effect under non-inductive conditions that should be taken into consideration while choosing a promoter for the experiment.