USTC/HybridOperator

From 2007.igem.org

Naming Rules for Oxx and Oxy below

As we mentioned in Operator Composition, biologists always use symmetric operators like O22 because they are the most suitable ones to be bound to by their own specific repressor(s).

Hybrid Operators for Heterodimeric Repressor

Assumed that R4 is the highly specific repressor protein that well binds to O44, and so does R2 to O22, well, then it is clear that asymmetric O24 can be bound to both by R2 and R4. However, neither of them is very suitable because R4 is highly specific only for O44 rather than O22, and vice versa. Luckily these LacI-family Repressors are also symmetric, each of them is combined by two same monomers, so one side of R2 can bind to O24's left-half strand while the other side would not bring about any remarkable effect on the right. To bind to half strand could still maintain repression, but distinctly it could no longer be that strong. Now we can say that hybrid operator O24 is a “weaker” operator for R2 or R4 because their weaker binding compared with R2+O22 or R4+O44. Furthermore, the dimerization region of our artificial repressors are same, so they can combine into the heterodimeric form, for example, heterodimer R2.R4, one side of R2 can bind to O24's left-half strand while the other side of R4 bind on the right half as well.

Supposed mechanism of hybrid operator. It appears as a weak operator to homodimers, and only heterodimers can bind to it tightly.

Hybrid Operators for Weaker Binding

There is another kind of “weaker” operators Ox6, for example, AATTGTGAAC GCTCATAATT (O46). The symmetric prototype AATTATGAGC GCTCATAATT (O66) is an interesting operator, because all the known LacI-family repressors would not have detectable repression on it [1]. It is obvious that O46 is a “weaker” operator only to R4 (compared with R4+O44), but not a valid operator to other repressors that are not specific for O44. On the other hand, O24 is a “weaker” operator to both R4 and R2. We also try to use random sequence GACGACTGTA TACAGTCGTC (O77) to replace O66, that is to say, O47 is sequence AATTGTGAAC TACAGTCGTC. It seems that O47 might behave just like O46, but actually it is too weak to be bound to by R4 for our purpose. (Comparable data of Ox6 and Ox7 on page Operator Composition.)

References

1. Sartorius, J.; Lehming, N.; Kisters, B.; von Wilcken-Bergmann, B. & Müller-Hill, B. (1989), lac repressor mutants with double or triple exchanges in the recognition helix bind specifically to lac operator variants with multiple exchanges., EMBO J 8(4), 1265--1270.