Team:NTNU Trondheim/Description

Project Description

Background

Diabetes is a metabolic diseases of high level of blood sugar (glucose). People having diabetes can not produce insulin hormone enough to remove excess glucose from the blood. Type 1 diabetes is characterized by a lack of insulin production. Type 2 diabetes is caused by the body’s ineffective use of insulin and it often results from excess body weight and physical inactivity.

Nowadays, around 3% of the norwegian population and 400 million people worldwide suffer from diabetes.

Early diagnosis can be accomplished through blood testing.Treatment of diabetes involves lowering blood glucose and the levels of other known risk factors that damage blood vessels. Healthy lifestyle measures have been shown to be effective in preventing or delaying the onset of type 2 diabetes.

References: http://www.who.int/topics/diabetes_mellitus/.

Motivation

To create an efficient glucose sensing system in bacteria.
To create a system that can be encapsulated in alginate, using the capsules as a vector for deploying the bacteria for biomedical applications.
To find a simpler way than checking the blood sugar level several times a day.

Objectives

To engineer glucose sensing system in Pseudomonas putida by expressing red fluorescent protein mCherry upon its (glucose) detection (as a proof of concept).
To encapsulate bacteria in alginate and tailor capsule properties to suit best for our engineered cells and purposes.
To improve the Matchmaker website.
To develop a modeling of the glucose biosensor system.

Methods

Genetic engineering

Why did we choose to engineer Pseudomonas putida?

After extensive research we decided to choose Pseudomonas putida over E. coli as transforming organism. Whereas E.coli phosphorylates glucose during its uptake, P. putida …

Glucose metabolism in Pseudomonas putida

Glucose, gluconate and 2-ketogluconate are transported into the cell without being modified (see Figure 1). In the periplasm, glucose can either be transformed into gluconate and subsequently to 2-ketogluconate or it can pass the inner membrane. Approximately half of glucose in the periplasm is directly channeled into the cytosol. The other half is processed via the 2-Ketogluconate peripheral pathway (less than 10% of gluconate produced by glucose is directly passing the inner membrane (del Castillo 2007)). In the cytosol, glucose is transformed via glucokinase (glk) and glucose-6-phosphate 1-dehydrogenase (zfw) into 6-phosphogluconate. 2-Ketogluconate is modified by 2-ketogluconate kinase (kguK) and reductase (kguD), which results in 6-phosphogluconate as well. Finally, 6-phosphogluconate is transformed into 2-keto-3-deoxy-6P-gluconate and subsequently cleaved into pyruvate and glyceraldehyde-3-phosphate (Daddaoua 2010).