Difference between revisions of "Part:BBa K3019001"

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	The results are as expected, negative control does not show almost any fluorescence. The promoter shows fluorescence higher than the positive control.		The results are as expected, negative control does not show almost any fluorescence. The promoter shows fluorescence higher than the positive control.

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Revision as of 21:14, 17 October 2019

pRNR1 promoter from S. cerevisiae

1000bp up the RNR1 gene. Constitutive promoter.

Sequence and Features

INTRODUCTION

In order to determine the strength of pRNR1 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	pRNR1	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 pRNR1-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

The results are as expected, negative control does not show almost any fluorescence. The promoter shows fluorescence higher than the positive control.