Part:BBa_K122000

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

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.

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 pPGK1-EGFP-tCYC1	pPGK1	EGFP	tCYC1	pRS306
2	pRS306 pTDH3-EGFP-tCYC1	pTDH3	EGFP	tCYC1	pRS306
3	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
Positive control(pTDH3)	DOM90	MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]	pRS306 pTDH3-EGFP-tCYC1
pPGK1	DOM90	MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]	pRS306 pPGK1-EGFP-tCYC1

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 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.

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

Negative control	Positive control	pPGK1		CSM
Colony 1 Replicate 1	Colony 1 Replicate 1	Colony 1 Replicate 1	Colony 2 Replicate 1
Colony 1 Replicate 2	Colony 1 Replicate 2	Colony 1 Replicate 2	Colony 2 Replicate 2
Colony 1 Replicate 3	Colony 1 Replicate 3	Colony 1 Replicate 3	Colony 2 Replicate 3
Colony 1 Replicate 4	Colony 1 Replicate 4	Colony 1 Replicate 4	Colony 2 Replicate 4
Colony 1 Replicate 5	Colony 1 Replicate 5	Colony 1 Replicate 5	Colony 2 Replicate 5
Colony 1 Replicate 6	Colony 1 Replicate 6	Colony 1 Replicate 6	Colony 2 Replicate 6
Colony 1 Replicate 7	Colony 1 Replicate 7	Colony 1 Replicate 7	Colony 2 Replicate 7
Colony 1 Replicate 8	Colony 1 Replicate 8	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.

CONCLUSIONS

<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.