Team:ESBS-Strasbourg/measurements

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In general

• BD means the four DNA binding proteins: LexA, LacI, CI and TetR
• The promoter consists of a repeat of the binding sequence of the DBP and the minimal Promoter (containing TATA box and the Kozak sequence)
• There is a glycin linker between every protein fused
• The IRES also contains already a Kozak sequence
• The used colors are
  • Blue for the self activator
  • Yellow for the cross activator
  • Red for the (cross) repressor
    

Activity and functionality of the single activator constructs

Here we are going to test two things:

• The functionality of the inducer construct in regard to the position of the XFP. This will first be tested for one construct and then later on be transfered to the others. The thing to measure is the amount of CFP under same inducer conditions for the three constructs.
• The strength of the promoters for the different DNA Binding Proteins. Therefore we will induce our system with different time steps and measure the outcome of the CFP. Then we'll try to find couples of DBP with similar activities/Kds by changing the number of operons. The thing to measure is the ratio between YFP and CFP under different inducer conditions and different operon numbers and the total amount of CFP.
  

1.1 Inducer

1.2 Reporter construct

Stability of the self activator

Here we are going to test if the self activation of the constructs is sufficiently strong to keep the signal through several cell divisions. The thing to measure is the CFP amount after induction and after several (~10) cell divisions.
2.1 Inducer

Same as under 1.1

2.2 Reporter construct

Effect of the repressor

Here we can measure two things:

• the efficiency of the repressor. The thing to measure is the CFP amount.
• the efficiency of the IRES. The thing to measure is the ratio between YFP and RFP.
  

3.1 Inducer

3.1 Reporter construct

Same as under 1.1

Tag-testing

Here first of all the efficiency of our tags are tested with XFP before we can test out at which position to put them into the BD-VP16-XFP-NLS-PEST-APC construct. The thing to measure is the development of the XFP.