Revision as of 20:31, 21 September 2011

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Lysis Selection System

Lysis selection system Main | Lysis Characterization | DNA Recovery | DNA Selection | T7 Promoter Variants

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):

Parts Registry Number	Strength	Mutation Position	Sequence
[http://partsregistry.org/Part:BBa_K613001 K613001]	Wildtype	None	TAATACGACTCACTATAGGGAGA
[http://partsregistry.org/Part:BBa_K613002 K613002]	.14	-16	TGATACGACTCACTATAGGGAGA
[http://partsregistry.org/Part:BBa_K613003 K613003]	.3	-2	TAATACGACTCACTACAGGGAGA
[http://partsregistry.org/Part:BBa_K613004 K613004]	.54	-17	CAATACGACTCACTATAGGGAGA
[http://partsregistry.org/Part:BBa_K613005 K613005]	.8	3	TAATACGACTCACTATAGGGTGA
[http://partsregistry.org/Part:BBa_K613006 K613006]	1.11	3	TAATACGACTCACTATAGGGCGA

Variants with additional lac operator (LacI repressed):

Parts Registry Number	Strength	Mutation Position	Sequence
[http://partsregistry.org/Part:BBa_K613007 K613007]	Wildtype	None	TAATACGACTCACTATAGGGAGAGGAATTGTGAGCGGATAACAATT
[http://partsregistry.org/Part:BBa_K613008 K613008]	.14	-16	TGATACGACTCACTATAGGGAGAGGAATTGTGAGCGGATAACAATT
[http://partsregistry.org/Part:BBa_K613009 K613009]	.3	-2	TAATACGACTCACTACAGGGAGAGGAATTGTGAGCGGATAACAATT
[http://partsregistry.org/Part:BBa_K613010 K613010]	.54	-17	CAATACGACTCACTATAGGGAGAGGAATTGTGAGCGGATAACAATT
[http://partsregistry.org/Part:BBa_K613011 K613011]	.8	3	TAATACGACTCACTATAGGGTGAGGAATTGTGAGCGGATAACAATT
[http://partsregistry.org/Part:BBa_K613012 K613012]	1.11	3	TAATACGACTCACTATAGGGCGAGGAATTGTGAGCGGATAACAATT

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.

Team:EPF-Lausanne/Our Project/T7 promoter variants/t7prom

From 2011.igem.org