Team:BOKU-Vienna/Basic Part
Basic Part
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.
This promoter is build up fully modular. It starts with a GAL4 binding site derived from GAL1. After this transcription factor binding site a neutral spacer is placed to avoid steric hindrances between GAL4 and the TATA-box-binding-proteins of the preinitiation complex. Following the TATA-box there is the core1 consisting of 30 random nucleotides. It functions as a spacer between the TATA-box and the TSS sequence. In yeast the minimal gap between TATA-box and TSS should be 30 nucleotides. TSS, core1 and TATA-box are the minimal elements for an active promoter but without an additional activation element (in this case the GAL4 binding site) the activity of the minimal promoter would be to low for efficient protein expression.
All information taken from Redden 2015.
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 synGAL promoter combined with a Kozak sequnce showed the highest expression levels of GFP. It is about twice compared to the synGAL 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.
A: fully induced pGAL with Kozak sequence compared to the wildtype
B: non induced pGAL with Kozak sequence compared to fully induced one
C: pGAL with Kozak sequnece (sample B3 36), pGAL without Kozak sequence (sample D4 37), pTDH3 and wildtype.
