Team:PennState/diauxie/intro

Diauxie Elimination

Introduction

Implementation

Progress

Parts

References

NHR
Biosensors

Introduction

Smart Fold

Overview

Nuclear Fusion

Overview

There are currently a only few dependable transcriptional induction systems that are readily available for use in E. coli and other bacteria. Some common systems use proteins such as LacI and AraC to sense the level of sugars such as lactose and arabinose respectively. In both these cases the presence of glucose affects the amount of transcription promoted by each operator; in wild-type bacteria the genes downstream of these operators are involved with metabolism of the sugar associated with the control of that gene system. This phenomenon of preferential sugar usage for cell growth is called diauxie.

In this project our goal is to introduce a new induction system that uses xylose as the inducer. To make this system unique, transcription will be activated in the presence of xylose regardless of the presence of glucose in the cell. This will create a novel and useful new way of initiating transcription that isn’t limited to cases where glucose is not a carbon source.

In addition to creating a valuable new tool for the part registry, this project has useful applications in the bioproduction industry, including (but not limited to) conversion of biomass to ethanol using bacteria. Common biomass feedstocks used for production contain a 50:50 mixture of glucose and xylose. When cells are grown on this mixed carbon source, diauxie causes glucose to be used first, then depending on the process xylose is consumed or removed as waste. This leads to inefficiency in production, especially if a continuous process is desired. Separation of xylose from glucose is very costly, and so is the lag time when the cells are switching from glucose to xylose metabolism. The induction system we are creating would eliminate this problem by eliminating diaxuie, leading to more efficient production by using both sugars at the same time.

The System

In wild-type E. coli the xyl operon controls xylose transport and metabolism.

• The left facing xylAB genes code for xylose isomerase and kinase.
• The other set of genes, xylFGH are right facing and code for active xylose transport proteins.
• A bidirectional operator is necessary because of the opposite directions of transcriptional control for xylAB and xylFGH.
• XylR is located downstream of xylH but is controlled by its own weak promoter and is involved in regulation. Transcription is initiated when the protein XylR binds to xylose; this complex then binds to the XylR binding site located at both ends of the operator.

The system also requires binding of the activated complex cAMP-CRP (Cyclic adenosine monophosphate - cAMP Receptor Protein) to the individual CRP binding site to activate transcription. In terms of circuits, XylR and cAMP-CRP are the two inputs for this “and” logic gate. The protein cAMP is an indicator of the glucose concentration in the cell and becomes more abundant with glucose depletion. Another protein called XylE is a passive xylose transporter and exists elsewhere in the E. coli chromosome. Over expression of the xylE gene may help xylose enter the cell and begin its metabolism cycle.

Natural Operation - PN plasmid (see Implementation)

The presence of xylose and the absence of glucose are required for natural transcriptional activation in the xyl operon. Our goal was to have activation only dependent on the presence of xylose, independently of glucose.