Difference between revisions of "Part:BBa K105031"

Revision as of 07:31, 9 October 2019 (view source) Valeriia (Talk | contribs) ← Older edit		Revision as of 07:33, 9 October 2019 (view source) Valeriia (Talk | contribs) Newer edit →
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	|2		|2
−	|pRS306 pRPL18B-EGFP-tCYC1	+	|pRS306 pCYC16-EGFP-tCYC1
	|CYC16		|CYC16
	|EGFP		|EGFP

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Revision as of 07:33, 9 October 2019

cyc16 minimal promoter

This is a derivate of BBa_K105027. Due to a point mutation in the TATA box region the efficiency of this minimal promoter is only 16 % of the natural yeast CYC1 promoter.

Sequence and Features

ITRODUCTION

In order to determine the strength of pCYC16 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.

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 pTDH3-EGFP-tCYC1	TDH3	EGFP	tCYC1	pRS306
2	pRS306 pCYC16-EGFP-tCYC1	CYC16	EGFP	tCYC1	pRS306

After construction of plasmids was finished, we have transformed S. cerevisiaeDOM90 (leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG) strain with plasmids listed above to create the strains (table 2).

Table 2. S. cerevisiae strains created.

	Strain name	Genotype	Plasmid integrated
Positive control	DOM90	MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]	pRS306
Negative control	DOM90	MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]	pRS306
Test	DOM90	MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]	pRS306

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 0.1 to 0.9 and distributed to 96 well plate (clear flat bottom). 2 replicates of 4 colonies from each 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	pCYC16
Colony 1 Replicate 1
Colony 1 Replicate 2
Colony 2 Replicate 1
Colony 2 Replicate 2
Colony 3 Replicate 1
Colony 3 Replicate 2
Colony 4 Replicate 1
Colony 4 Replicate 2

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.