Synthetic Biology is a new area of Bioengineering research that combines Science and Engineering in order to design and build novel biological functions. The main goal of this discipline is to build up the desired functions through a modular design, in which a set of well characterized biological parts (e.g. promoters, genes, transcriptional terminators, etc.) is used. In the last five years, a collection of biological parts (Registry of Standard Biological Parts) has been developed by the Massachusetts Institute of Technology (MIT). These parts, called BioBrick parts, can be defined "standard" because their structural features allow an easy assembly of different components through a standard methodology, based on special kind of plasmids containing a cloning site with four restriction sites, two of which are isocaudamers. In this way, it is possible to construct user-defined gene regulatory networks that implement novel biological functions using basic or composite BioBrick parts and to incorporate these functions in the desired organisms, in order to allow them to exhibit functions that normally they do not exhibit. The design and validation of novel artificial gene networks finds applications in several areas, like medicine (detection and destruction of tumor cells, novel clinical tests, regenerative medicine, etc.), pharmaceutics (construction of organisms able to perform a large scale production of rare plants-derived drugs), renewable energy (construction of microorganisms able to synthesize biofuels), electronics and automation (implementation of sensors, actuators, amplifiers, logic functions, programmable waveform generators, etc.).

The Registry of Standard Biological Parts is also the basis of the international Genetically Engineered Machine (iGEM) competition, at which student and researchers of the universities worldwide can take part: in this contest, in May each participant group receives the whole set of available BioBrick parts of the Registry and can use them to assemble new original synthetic biological systems, which must be documented and presented at MIT in November. The main goal of this competition is to promote standardization in Synthetic Biology and to realize new BioBrick parts that can upgrade the Registry collection.

All the activities in Synthetic Biology field are performed in collaboration with the CIT (Centro interdipartimentale di Ingegneria Tissutale).

Current project activities

Selection and engineering of microbial strains for biofuel production
Cheese whey is classified as a special waste for is high biochemical and chemical oxygen demand. Even if whey can be valorized by extracting high value substances, like whey-proteins, at the end of the treatment the residual liquid, called whey permeate, is still a special waste for its high lactose content (about 45 g/L). A possible approach for permeate disposal is to convert its pollutant nutritional load into a precious biofuel, fermenting lactose into ethanol. Many microorganisms can efficiently metabolize lactose and many other organisms are able to ferment sugars into ethanol. Unfortunately, no naturally occurring organism is able to ferment lactose into ethanol with high yield. Our research focuses on the selection of promising microorganisms able to perform this transformation and the optimization of this process through Synthetic Biology principles.
Up to date, we have engineered E. coli bacterium engineered to convert efficiently lactose into ethanol, a precious biofuel. Three main enzymes have been involved in this transformation: beta-galactosidase, pyruvate-decarboxylase and alcohol-dehydrogenase II. Beta-galactosidase (native bacterial lacZ gene) has been over-expressed to obtain higher lactose-glucose conversion yield and to avoid the negative feedback caused by glucose in wild type lac operon. Coding sequences of pyruvate-decarboxylase (pdc) and alcohol-dehydrogenase II (adhB) from Zymomonas mobilis bacterium, essential in alcoholic fermentation pathway, have been designed by DNA chemical synthesis and codon-optimized for E. coli. The final circuit includes the device to metabolize lactose and the ethanol-producing operon, containing pdc and adhB. Finally, several well-characterized promoters have been used in order to optimize the expression of the three enzymes and to limit the metabolic burden of this artificial gene network.

Definition and validation of new methods for standard parts characterization
The Registry of Standard Biological Parts is a continuously upgraded archive that contains over 4000 parts up to date. Although the BioBrick parts conform to a physical assembly standard, the definition of new standard measurement methods are needed in order to allow the quantitative characterization of these parts. The application of standard measurement methods would allow to consider the Registry as a real biological parts user-handbook, in which the quantitative characteristics of the parts are reported. A large part of our activities in this field are focused on the development and improvement of new model-based approaches for promoters characterization. Promoters are DNA sequences able to trigger the expression of the downstream genes. Several efforts must be performed to characterize the strength, stability, metabolic burden and optimal working conditions of constitutive and inducible promoters. Our activities consist in:

1. the design and construction of suitable gene networks for promoter characterization using BioBrick standard DNA cloning methodologies, mainly using reporter genes encoding fluorescent proteins (e.g. GFP - Green Fluorescent Protein) and
2. the quantitative validation of standard measurement methods with a high-throughput approach, mainly using a microplate reader as measurement system, able to incubate the microorganisms bearing the gene networks in their optimal environmental conditions, to measure the cell growth rate and to measure the fluorescence of the reporter genes.

An optimized version of the Relative Promoter Units (RPU) approach, proposed by Kelly et al., 2008, has been applied to characterize the strength of 3 constitutive promoters and 5 inducible devices up to now.

iGEM competition
Since 2008 the University of Pavia takes parto to the iGEM competition, recruiting new students from engineering/biology/biotechnology courses every year, developing an original Synthetic Biology project from May to November and presenting it at MIT.
Past projects have been awarded:

1. Engineering E. coli to multiplex and demultiplex signals (http://2008.igem.org): Silver Medal in 2008.
2. Ethanol? Whey not! (http://2009.igem.org): Gold Medal and Best Food or Energy Project in 2009.

 

People working on the topic:
Lorenzo Pasotti, Susanna Zucca, Paolo Magni

Sponsor