Team:Utah State/Introduction

The aim of the Utah State University iGEM project is to develop improved upstream and downstream processing strategies for manufacturing cellular products using the standardized BioBrick system. First, we altered the broad-host range vector pRL1383a to comply with BioBrick standards and enable use of BioBrick constructs in organisms like Pseudomonas putida, Rhodobacter sphaeroides, and Synechocystis PCC6803. This vector will facilitate exploitation of advantageous characteristics of these organisms, such as photosynthetic carbon assimilation. Following expression, product recovery poses a difficult and expensive challenge. Downstream processing of cellular compounds, like polyhydroxyalkanoates (PHAs), commonly represents more than half of the total production expense. To counter this problem, secretion-promoting BioBrick devices were constructed through genetic fusion of signal peptides with protein-coding regions. To demonstrate this, the secretion of PHA granule-associated proteins and their affinity to PHA was investigated. Project success will facilitate expression and recovery of BioBrick-coded products in multiple organisms.

Since the beginning of iGEM, BioBricks have chiefly been designed for use in E.coli. This has primarily been due to the efficient growth rate of E.coli and its relatively thorough characterization. However, the employment of the BioBrick system in host organisms other than E. coli would greatly enhance and expand the field of synthetic biology. In order to investigate the BioBrick system in other organisms, it is imperative that a reliable broad-host-range vector be developed. The 2009 Utah State iGEM team is building on the 2008 University of Hawaii team’s efforts to develop a broad-host-range BioBrick vector that would make possible the use of BioBrick parts, devices, and systems in organisms other than E. coli. The organisms under investigation are Pseudomonas putida KT2440, Synechocystis PCC 6803, and Rhodobacter sphaeroides. Additionally, our project seeks to break borders in another way: through the construction of Silver-fusion compatible BioBrick parts for secretion-based recovery of recombinant proteins and other compounds, like polyhydroxyalkanoates.

One of the BioBrick borders we seek to break is that of Pseudomonas putida. This bacterium would open the BioBrick doors to soil applications. Pseudomonas putida is a non-pathogenic, gram-negative soil bacterium with optimal growth at room temperature. The diversity of its metabolic pathways allows it to be used for bioremediation purposes; it can degrade many polluting aromatic hydrocarbons including toluene, benzene, xylene, naphthalene, and styrene. This organism can also act as a biocontrol agent (Lemanceau, 1992; Haas and Defago, 2005), suppressing the growth of fungi. P. putida performs these functions while colonizing the rhizosphere of plant roots, enhancing the growth of the plant through these and other means (Albert and Anderson, 1987; Bakker et al., 1986). The genome of P. putida KT2440 has been sequenced, allowing more extensive genetic analyses and contributing to this strain being the “preferred host for cloning and gene expression for Gram-negative soil bacteria” (Nelson et al., 2002). Potential applications include BioBrick devices for enhancing the catabolism of environmental pollutants, the implementation of BioBrick devices used to protect plants (and its subsequent consumers) against pathogens not previously defended against, and the use of BioBrick devices to increase crop yields.

Another BioBrick border we'd like to break is that of cyanobacteria. We have specifically been working with Synechocystis PCC 6803. This bacterium would allow BioBricks to be used in photosynthetic applications. Synechocystis PCC 6803 is a Gram-negative bacterium that can produce energy either through photosynthesis or respiration (Tabei et al., 2007). It also displays a circadian rhythm in several of its cellular functions (Kucho et al., 2005) and can take up foreign DNA (Williams 1988). It can also grow in a variety of temperatures (Gombos et al., 1992). Cyanobacteria in general play an important role in nitrogen fixation for crops and are a major player in rice cultivation (Irisarri et al., 2001). Potential applications include the use of BioBrick devices in bioenergy, wastewater treatment, crop yields, and biomanufacturing processes that take advantage of the fact that a carbon source is not needed.

A third border that we aim to break is that of Rhodobacter sphaeroides, an organism usually found in the anaerobic mud of ponds and lakes where there is access to sunlight. This is a very metabolically diverse organism that has potential for providing a myriad of BioBrick opportunities. Rhodobacter sphaeroides can grow under a variety of conditions: aerobic or anaerobic respiration, photosynthesis, and fermentation; it has optimal growth in microaerophilic surroundings. It can also fix dinitrogen as its sole nitrogen source (Mackenzie et al., 2007). Similar to E. coli, this organism moves with a single flagellum. R. sphaeroides has more membrane surface per cell than other organisms used to express membrane proteins, making it an ideal host for overexpressing and studying such proteins. It is capable of making biofuels through the process of lithotrophy (Roy et al., 2008) and other pathways (Yokoi et al., 2002). R. sphaeroides is also capable of tolerating and reducing at least 11 rare earth metal oxides and oxyanions, making it an excellent candidate for bioremediation and detoxification purposes (O’Gara et al., 1997). Many of the above-listed characteristics place R. sphaeroides in the spotlight for use in biomanufacturing. Possible BioBrick applications with R. sphaeroides include membrane protein studies (including secretion and protein overexpression studies), biomanufacturing, bioenergy, and bioremediation/detoxification.

To even further break down BioBrick borders, composite devices were constructed to investigate phasin and green fluorescent protein secretion. Secretion of phasin was studied to show that these PHA-associated proteins are targetable for export out of the cytoplasm, and that optimization of phasin expression and binding may facilitate bioplastic secretion. Constructs for GFP translocation were made in parallel with the phasin secretion devices. These GFP constructs provide a visually or spectrofluorometrically detectable control due to a high level of fluorescent protein accumulation. Successful GFP translocation would reinforce the potential of phasin export, which is not as readily monitored. Beyond the scope of this project, the constructed signal peptides and GFP BioBricks can readily be used by other researchers for recombinant protein secretion studies.

The overall goal of this project is to demonstrate the concept of “BioBricks without Borders” by expanding the use of broad-host vectors for expression of BioBricks in multiple organisms and by demonstrating secretion for simplified recovery of recombinant proteins using BioBrick constructs. More specific goals of this project are to:

• Determine how broad-host range vectors can be modified to comply with the BioBrick assembly standard.
• Use broad-host range vectors to transform Synechocystis PCC6803, R. sphaeroides, and P. putida by triparental mating.
• Create a BioBrick genetic library of Silver fusion-compatible signal peptides and coding regions for secretion studies.
• Test the functionality of BioBrick devices and determine methods for detecting phasin and/or PHA secretion.

The following sections provide more extensive details about these goals, experimentation and testing, and the results and conclusions from this project.