BBa_K2294007

BBa_K2294007 is a short synthtetic promoter which can be induced by galactose. This promoter contains already a Kozak sequence for the expression of proteins. The promoter is only 160 bp long.

Characterization

For characterization the synGAL promoter was combined with GFP and the CYC1 terminator and afterwards integrated into the ura3 gene of Saccharomyces cerevisiae. The GFP fluorescence was measured using a flow cytometer after inducing with 3 different galactose concentrations (0.5%, 1% and 2%). Every condition (except of non induced and the wildtype) were cultivated in triplicates.

The preculture was performed in 1 mL selective YPD. Prior to main culture inoculation the cells were washed twice in YP without any carbon source to remove remaining glucose. 100µL of the preculture were used for inoculation of 2 mL main culture which was cultivated at 30 °C at 280 rpm for around 5 hours. As controls a wildtype and a clone expressing GFP under the constituve pTDH3 promoter were cultivated. In addition, the synGAL promoter without a Kozak sequence was investigated. Before flow cytometer measurement cells were washed in PBS twice and afterwards diluted to a final OD of 0.5.

There is no significant difference between the different galactose concentrations which were investigated. The galpromoter combined with a Kozak sequnce showed the highes expression levels of GFP. It is about twice compared to the gal promoter without a Kozak sequence and three times higher compared to the pTDH3 promoter of Saccharomyces cerevisiae. The clones expressing GFP under the control of the consitutive TDH3 promoter were cultivated in YP + 2% glucose.