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Lysis Selection System

T7 Promoter Variants

Introduction

The success of any piece of biological circuitry, like the kind we are presenting in our system, hinges substantially on the time-scales required for the activation of its components. If one component reacts more quickly than another, the whole system will desynchronize and all pertinent biological information will be lost.

In our case, one of the key steps is the activation of the lysis cassette which is controlled by the T7 promoter. The rest of the system -- including any reporter systems that would lead to T7 RNA polymerase binding to a T7 promoter -- may depend on other parameters that affect the timescales in very delicate ways. It becomes crucial to have a variety of T7 promoter variants on hand, each of which has different strengths and efficiencies in order to accomodate the various system time-scales.

We made two families of T7 promoter variants. Each family has six designed mutants and three randomer mutants. The only difference between the two families is the addition of a lac operator sequence downstream of the T7 promoter. These promoter variants were characterized using fluorescence, revealing a wide range of promoter efficiencies.

List and Sequences of Variants

Non-random T7 promoter variants

The T7 promoter variants in this section were designed based on considerations from literature. These sequences, and their corresponding predicted strengths, also come from literature. The

Variants without additional lac operator (constitutive):

Variants with additional lac operator (LacI repressed):

Randomer T7 Promoter Variants

The randomer variants were made by ordering random mutants at the following positions with respect to the consensus logo for the T7 promoter:

• -7-12
• +1+3, -1-3
• -10

These were produced both for the T7 and the T7-lac promoter.

Characterization of Designed Variants

Characterization of Randomer Variants