Team:Utah State/Project

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.

need an intro paragraph about broad host vectors, etc.

BioBricks were constructed to investigate phasin and green fluorescent protein secretion to provide proof-of-concept. 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.

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This is where all of the text about secretion goes - First talk about secretion in general and why we care (downstream processing, etc.); next talk about what exactly we are looking at as an example (phasin/bioplastic secretion, along with GFP as an easier-to-monitor example);

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Conclusions go here

• This is where references go