Construction Of A Library Of Yeast S. Cerevisiae Synthetic Promoters Activated By Orthogonal Chimeric Proteins

Synthetic biology is a multidisciplinary subject between engineering and molecular biology.Currently,building biosensors is one of the hot research fields in synthetic biology.A biosensor is a DNA-based device that detects a chemical in the cellular environment and responds to its presence by either emitting an output signal such as fluorescence or triggering a pathway that leads,for instance,to the chemical degradation.It is apparent that biosensors have a vast application range,from removal of pollutants to sick cell detection.In this project,we both built and optimized ?-estradiol biosensors for the yeast S.cerevisiae.At the beginning,we optimized our biosensors by using the common ways mentioned above.We utilized constitutive promoters with different strengths in the sensor section(from the strong p GPD and p TEF2 to the rather weak DEG1t_p CY1 no TATA),and we replaced truncated_p CYC1 min with p CYC1 min and p CYC1core(both weak)in the reporter section—i.e.we did not change only the 7lex2 Op cassette.Furthermore,various activation domains(VP16,B42 and m DR521-805)were taken into account to build variants of the chimeric activator.We constructed nine yeast strains and tested them with ?-estradiol induction experiments.We found out that by using a strong promoter in sensor section or a strong activation domain as a component of the activator,the system sensitivity would increase,while the tolerance would decrease.However,no significant difference in terms of signal separation was detected.Furthermore,we studied how does the position of Lex A operator effect.We inserted three lex Op2 operators inside the sequence of p CYC1 core.Two adjacent lex2 Op were separated by 2 nt,6 nt,9 nt,15 nt,21 nt,and 42 nt.These new synthetic promoters were inserted into reporter section and tested with ?-estradiol induction experiments in a yeast biosensor system.From our results,we concluded that the best distance between lex Op2 operators(i.e.the one that corresponds to the maximal signal separation without altering the system sensitivity and tolerance)should be between 6 and 9 nt.Also we inserted a single operator at 6 nt,10 nt,37 nt,and 60 nt from the main TATA box of p CYC1 core.Experiments based on ?-estradiol induction(similar to those in the first group)were carried out.From our results,we concluded that the best distance between lex Op2 operator and the TATA box is 37 nt.On the whole,the goal of this project was to find out the way the position of lex Op2 operator,within a synthetic promoter,shapes the performance of ?-estradiol biosensors.Through a series of synthetic promoter designs and tests,we can conclude that the working of our Lex A-based biosensor systems is optimized when the distance between adjacent lex Op2 operators is between 6 and 9 nt and the distance between the TATA box and the nearest lex Op2 operator is 37 nt.