A simple method for incorporating sequence information into directed evolution experiments
1. A simple method for incorporating sequence information into directed evolution experiments Kyle L. Jensen*, Hal Alper*, Curt Fischer, Gregory Stephanopoulos Department of Chemical Engineering Massachusetts Institute of Technology sequence phenotype
2.
3. Here, a P Ltet promoter was mutated to create a library of promoter variants Alper H., C. Fischer, E. Nevoigt, and G. Stephanopoulos, 2005. Tuning genetic control through promoter engineering. Proc. Natl. Acad. Sci. U S A 102:12678-83.
5. The 69 promoter variants spanned an 800-fold range of activity - How different are the underlying, mutagenized sequences? - What, on a sequence level, causes the variation? 800 fold range Log relative fluorescence Mutant number Top 50% Bottom 50%
6. Each of the 69 mutants had a unique sequence and incorporated multiple transition SNPs mutations promoter region Log relative fluorescence Mutant number Position [nt] Mutant number
7.
8. Each individual position can be evaluated using a simple binomial distribution Same as: what's the probability of getting heads 14 of 20 coin tosses? P-value: 14 or more heads out of 20 Assuming the positions are independent Position [nt] Class distribution
9. Similar analysis over the promoter region revealed 7 positions significantly correlated with activity Class distribution Position [nt]
10. Position [nt] Mutant number Class distribution Log relative fluorescence Mutant number Position [nt]
11. A similar analysis can be applied to an arbitrary number of mutants and phenotypic classes 1 2 M . . . mutants M phenotypes Mutants with mutations as “position 35” . . . . . . . . . or 1 2 3 4 5 6 Y
12.
13. In our case, we tested 8 locations, spanning a range of functions & confidences Class distribution Position [nt]
14. 7/8 of the single position mutants were in agreement with the predicted function
15.
16. In summary, this simple method, based on multinomial statistics, can be used to link sequence variations to particular phenotypes