DEVELOPMENT OF A REPORTER METHOD FOR DETERMINING THE TIME OF INDUCTION IN ESCHERICHIA COLI WHEN USING AUTOINDUCTION MEDIUM

Abstract

A wide variety of proteins can be expressed in E. coli using inducible expression systems, such as the pET expression system in which T7 RNA polymerase transcribes coding sequences under the control of the T71ac promoter. This induction system works nicely with the auto-induction method as the amount of protein expression can be controlled by the availability of lactose to the culture. Investigation of protein expression revealed that culture aeration levels, medium composition and LacI levels affect the induction timing of cultures grown in auto-induction medium. To alleviate these issues, a self reporting expression system was designed using the fluorescent protein reporter, mCherry. This reporter was placed on the same message downstream of target proteins. To eliminate manual fluorescence measurements an automated fluorescent sensing instrument (Firefly) was designed by Buglab LLC and used for determining when mCherry and target proteins were expressed, eliminating the induction timing concerns.