Difference between revisions of "Part:BBa K165000"
Line 17: Line 17:
 
<partinfo>BBa_K165000 parameters</partinfo>
 
<partinfo>BBa_K165000 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
ITRODUCTION
 +
<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><p>MATERIAL AND METHOD
 +
</p><p>Plasmids used to conduct the experiment are listed in table 1.
 +
 +
Table 1. Plasmids created
 +
{| class="wikitable"
 +
|colspan="2"|
 +
|colspan="3"|Insert
 +
|
 +
|-
 +
|number
 +
|Plasmid name
 +
|Promoter
 +
|Gene
 +
|Terminator
 +
|backbone
 +
|-
 +
|1
 +
|pRS306 pTDH3-EGFP-tCYC1
 +
|TDH3
 +
|EGFP
 +
|tCYC1
 +
|pRS306
 +
|-
 +
|2
 +
|pRS306 pRPL18B-EGFP-tCYC1
 +
|MET25
 +
|EGFP
 +
|tCYC1
 +
|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).
 +
 +
Table 2. ''S. cerevisiae'' strains created.
 +
{| class="wikitable"
 +
|
 +
|Strain name
 +
|Genotype
 +
|Plasmid integrated
 +
|-
 +
|Positive control
 +
|
 +
|
 +
|
 +
|-
 +
|Negative control
 +
|
 +
|
 +
|pRS306
 +
|-
 +
|Test
 +
|
 +
|
 +
|
 +
|}
 +
 +
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.
 +
 +
Table 3. The 96 well plate layout for OD600 and Fluorescence measurements.
 +
{| class="wikitable"
 +
|
 +
|Negative control
 +
|Positive control(pTDH3)
 +
|MET25
 +
|-
 +
|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.
 +
 +
<h1>Image Demo</h1>
 +
  <p>
 +
    <img src = "MET25.jpg"
 +
        alt = "Picture of a happy monkey" />

Revision as of 06:06, 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. 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.

MATERIAL AND METHOD

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 pTDH3-EGFP-tCYC1 TDH3 EGFP tCYC1 pRS306
2 pRS306 pRPL18B-EGFP-tCYC1 MET25 EGFP tCYC1 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).

Table 2. S. cerevisiae strains created.

Strain name Genotype Plasmid integrated
Positive control
Negative control pRS306
Test

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.

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

Negative control Positive control(pTDH3) MET25
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.

Image Demo

 <p>
   <img src = "MET25.jpg"
alt = "Picture of a happy monkey" />