Team:Utah State/Project

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         <td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><a href="#abstract">Abstract</a><br />
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        <a href="#introduction">Introduction</a><br />
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        <a href="https://2009.igem.org/Team:Utah_State/FutureWork">Future Work</a><br />
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         <td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td>
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Why Break BioBrick Borders?
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      Since the beginning of iGEM, BioBricks have chiefly been designed for use in <i>E.coli</i>.  This has primarily been due to the efficient growth rate of <i>E.coli</i> and its relatively thorough characterization.  However, the employment of the BioBrick system in host organisms other than <i>E. coli</i> 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 <i>E. coli</i>.  The organisms under investigation are <i>Pseudomonas putida</i> KT2440, <i>Synechocystis</i> PCC 6803, and <i>Rhodobacter sphaeroides</i>. 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. </p>
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      One of the BioBrick borders we seek to break is that of <i>Pseudomonas putida</i>.  This bacterium would open the BioBrick doors to soil applications.  <i>Pseudomonas putida</i> 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.  <i>P. putida</i> 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 <i>P. putida</i> 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. </p>
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Another BioBrick border we'd like to break is that of cyanobacteria.  We have specifically been working with <i>Synechocystis</i> PCC 6803.  This bacterium would allow BioBricks to be used in photosynthetic applications.  <i>Synechocystis</i> 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.</p>
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<p class="class"> A third border that we aim to break is that of <i>Rhodobacter sphaeroides</i>, 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.  <i>Rhodobacter sphaeroides</i> 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.  <i>R. sphaeroides</i> 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).  <i>R. sphaeroides</i> 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 <i>R. sphaeroides</i> in the spotlight for use in biomanufacturing.  Possible BioBrick applications with <i>R. sphaeroides</i> include membrane protein studies (including secretion and protein overexpression studies), biomanufacturing, bioenergy, and bioremediation/detoxification.  </p>
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<p class="class">To <i>even further</i> 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.</p>
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Project Objectives
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<p class = "class">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:</p>
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<li>Determine how broad-host range vectors can be modified to comply with the BioBrick assembly standard.</li>
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<li>Use broad-host range vectors to transform <i>Synechocystis</i> PCC6803, <i>R. sphaeroides</i>, and <i>P. putida</i> by triparental mating. </li>
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<li>Create a BioBrick genetic library of Silver fusion-compatible signal peptides and coding regions for secretion studies.</li>
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<li>Test the functionality of BioBrick devices and determine methods for detecting phasin and/or PHA secretion.</li>
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<p class = "class">The following sections provide more extensive details about these goals, experimentation and testing, and the results and conclusions from this project.</p>
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Secretion: GFP, Phasins, and Bioplastics
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Recovery of cellular products is often a difficult and expensive challenge. As much as 80% of protein production costs are attributable to downstream processing (Hearn and Acosta, 2001). Likewise, the separation and purification cost for non-protein products, like polyhydroxyalkanaotes (PHAs) are significant and commonly represent more than half of the total process expense (Ling, 1998; Jung, 2005).  </p>
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Polyhydroxyalkanoates comprise a class of polyesters that are generated by a variety of microorganisms (Anderson and Dawes, 1990; Doi, 1990). These bioplastic compounds are intracellularly accumulated and stored as a reserve of carbon, energy, and reducing power in response to an environmental stress or nutrient limitation (Lee, 1996). Polyhydroxybutyrate (PHB) is the most common form of PHA. PHAs have comparable material properties to conventional plastics, like polypropylene, but are fully biodegradable and renewable (Steinbüchel and Füchtenbusch, 1998). As a result, PHAs are of particular interest as a sustainable source of non-petrochemically derived thermoplastics for use in an assortment of commercial and medical applications (Madison and Huisman, 1999).</p>
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<p class="class">Costs associated with the PHA manufacturing process have limited the widespread application of the bioplastic material (Lee, 1996). Economic analyses for industrial scale PHA production place the cost of PHAs at about $4-5/kg (Choi, 1997; Choi, 1999). In contrast, the average cost of petrochemically-derived plastic lies between $0.62-0.96/kg (Steinbüchel and Füchtenbusch, 1998). This significant discrepancy in expense is largely attributable to downstream processing. Traditional methods involving the use of solvents, enzymatic digestion, or mechanical disruption are expensive and impractical for industrial-scale recovery (Jung, 2005). As a result, the development of alternative methods for PHA recovery is necessary.</p>
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<p class="class">Genetic engineering strategies have been used in attempts to simplify PHA recovery and eliminate the need for mechanical or chemical cellular disruption. Jung et al. (2005) used recombinant E. coli MG1655 harboring PHA biosynthesis genes from C. necator to instigate spontaneous autolysis of the cell wall. Up to 80% of the cells in culture released PHA granules, which were subsequently recovered using centrifugation and washing with distilled H2O (Jung, 2005). Resch et al. (1998) used recombinant PHA-producing E. coli transformed with the E-lysis gene of bacteriophage PhiX174 from plasmid pSH2. Amorphous PHB in is pushed out of the cell through an E-lysis tunnel structure, which is an opening in the cell envelope (Resch, 1998). In this procedure, the osmotic pressure difference between the cytoplasm and the culture medium provides the driving force for PHA movement into the extracellular medium. The PHA is then recovered by centrifugation or through the addition of divalent cations (Resch, 1998). Although these methods use genetic means to bring about cellular disruption, these mechanisms still require cellular death and fail to promote a continuous production system.  </p>
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<p class="class">Recently, extracellular deposition of PHA granules was observed in a mutant strain of Alcanivorax borkumensis SK2, which is a marine bacterium that uses hydrocarbons as its source of carbon and energy (Sabirova, 2006). This finding by Sabirova et al (2006) is the first account of PHA accumulation outside of the cell (Prieto, 2007). However, the mechanism by which this deposition occurs is unknown (Sabirova, 2006; Prieto, 2007). A defined system for microbial excretion of PHAs has yet to be created. Such a system would be of value due to the potential to optimize and introduce the mechanism into other organisms with advantageous characteristics, such as fast-growing E. coli or photoautotrophic PHA-producers R. sphaeroides and Synechocystis PCC6803. </p>
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<p class="class">PHA-associated proteins, called phasins, strongly interact with the PHA granule surface (York, 2001; Maehara, 1999). Accordingly, PHA recovery may be possible by tagging the phasin protein for translocation. Specifically, the Silver fusion Biobrick standard can be used to create constructs in which a targeting signal peptide sequence is genetically fused to the phasin protein (Phillips, 2006). Fusing a signal peptide to a protein promotes export of the complex out of the cytoplasm (Choi, 2004; Mergulhão, 2005). The interaction of phasin with PHA is required for secretion-based granule recovery because PHA is a non-proteinaceous compound produced by the action of three enzymes (Suriyanmongkol 2007; Verlinden 2007). Consequently, the signal peptide cannot be directly attached PHA granules. The phasin protein with attached signal peptide binds to PHA granules, thereby creating a PHA-phasin-signal peptide complex that may be recognized by the cell for export. Figure X depicts this export process in general terms. Green fluorescent protein (GFP) translocation has been documented (Barrett, 2003; Santini, 2001; Thomas, 2001). Due to its ease of detection, studying GFP in parallel with phasin secretion mechanisms could provide a framework for determining the functionality of secretion systems.</p>
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Secretion-based product recovery mechanisms hold great potential to improve the economics of industrial-scale production systems. In addition to reduced downstream processing requirements, secretory production has additional benefits, such as potentially improved product stability and solubility (Mergulhão, 2005). Recombinant E. coli do not typically secrete high levels of proteins and functionality of proteins secretion is difficult to predict (Sandkvist, 1996; Choi, 2004). Accordingly, a trial-and-error approach with different combinations of signal peptides and promoters is recommended for any given protein, and will be discussed in more detail in subsequent sections (Choi, 2004).
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The functionality of protein secretion mechanisms is affected by the structural differences between gram-positive and gram-negative organisms (Desveaux, 2004; Sandkvist, 1996). Gram-positive species have a solitary cytoplasmic membrane, which effectively means that protein membrane translocation is equivalent to secretion in these species (Pugsley, 1993). Alternatively, gram-negative organisms have both an inner and outer membrane that proteins must cross for secretion.  Accordingly, proteins can either be exported into the periplasmic space or secreted fully into the extracellular medium (Pugsley, 1993). </p>
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There are five pathways observed for secretion of recombinant proteins in gram-negative prokaryotes, numbered I through V (Desvaux, 2004; Mergulhão, 2005). While all of these pathways differ mechanistically, they each promote secretion while maintaining the integrity of the cell structure (Koster, 2000). Types I and II are the most common pathways for recombinant protein secretion (Mergulhao, 2005) and will be discussed here. </p>
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<p class="class">Type I secretion is a single-step translocation of protein across both inner and outer membranes. (Binet, 1997). The constituents of this system include inner membrane proteins HlyB and HlyD, as well as the TolC outer membrane protein (Mergulhão, 2005; Desveax, 2004). These three proteins interact to form a channel that spans the periplasm (Mergulhão, 2005). Appending the last 42-60 amino acids of the HlyA protein C-terminus to the C-terminus of a recombinant protein targets the protein for secretion (Mergulhão, 2005; Gentschev, 2003; Hess, 1990). The HlyA signal sequence binds to the channel complex, resulting in ATP hydrolysis by HlyB to drive protein secretion (Gentschev, 2003). Proteins as large as 4000 amino acids can be secreted through the type I channel, which has an internal diameter of 3.5 nm and a length of 14 nm (Sapriel, 2003; Fernandez and de Lorenzo, 2001).  Unlike in the Type II pathway, the signal peptides of Type I secretion remain attached to the protein after export out of the cytoplasm (Blight and Holland, 1994).  Figure X depicts the secretion of a protein with a C-terminal fused HlyA signal peptide by Type I secretion. 
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The type II secretion pathway is a two-step process. The cytoplasmic protein must first be exported into the periplasm through the action of a translocase. Specifically, the Sec and Twin-arginine translocation (TAT) machinery facilitate protein movement across the inner membrane and will be discussed in detail in the next section.  After entering the periplasm, the protein can be translocated into the extracellular medium through the action of a secreton, which is a 12-16 core protein complex present in many gram-negative strains, such as E. coli K-12 (Cianciotto, 2005).  Although the secreton functionality is not completely understood, it is known that protein conformational changes are necessary for this process to be carried out (Mergulhão, 2005; Sandkvist, 2001).</p>
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Translocation of cellular products into the periplasm is advantageous over cytoplasmic production because recovery of periplasmic products is relatively simpler (Mergulhão, 2005). There are additional mechanisms for recovering periplasmic proteins if the secreton machinery is either not present in the host strain or incompatible with the protein of interest. These mechanisms are depicted in Figure X. L-form and Q-cells are mutant strains that have a weakened outer membrane, which allows for some proteins to leak into the extracellular medium (Mergulhão, 2005).  However, these organisms have reduced growth rates and are not ideal candidates for general cellular production.  The permeability of the outer membrane may be enhanced mechanically, such as by application of ultrasound, or through chemical treatment, such as through addition of Triton X-100 or 2% glycine (Kaderbhai, 1997; Choi, 2004). As another example, enzymatic digestion with lysozyme breaks the outer membrane to release periplasmic proteins (Shokri, 2003).  Yet another alternative involves coexpression of genes, such as kil, out, and tolAIII, that cause cellular lysis and subsequent release of recombinant proteins (Choi, 2004; Mergulhão, 2005). The downside to these alternatives is the weakening of cell integrity.
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Methods for Constructing BioBrick Parts
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One of the objectives of this project was to create a library of (link to silver fusion wiki) Silver-fusion compatible BioBrick signal peptides and protein-coding parts for secretion studies. The Silver-fusion assembly method was used because the standard BioBrick prefix and suffix do not facilitate fusion of two parts. The scar that forms from the overlap of compatible restriction enzyme sites XbaI and SpeI is not conducive to fusion because it contains a stop codon and is 8 nucleotides long. Because the scar is not a multiple of three, the sequence after the scar will be read out-of-frame. The Silver-fusion assembly method retains compatibility with the standard BioBrick assembly method, but fusion is allowed. A single nucleotide is removed from the prefix and suffix of Silver-fusion BioBricks so that the scar that forms from the ligation of XbaI and SpeI sites does not contain a stop codon and is 6 nucleotides in length.
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Five signal sequences were selected for this study based on the secretion pathway that they represent and their prominence in literature. The selected sequences are presented in Table X. Two protein coding regions were obtained: phasin and GFP. All of these sequences were designed for Silver-fusion compatibility. Four different promoters with an attached ribosome binding site were designed and then synthesized by DNA 2.0, followed by ligation into a BioBrick vector. Composite devices were assembled piecewise by cutting one part typically with EcoRI and XbaI, and the part to be inserted with EcoRI and SpeI. Analysis by PCR with the Primers VF2 and VR was used to qualitatively determine whether successful ligation had taken place. Once partially confirmed, samples were sequence at the Utah State University Center of Integrated Biotechnology.</p>
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To construct the OmpA, PelB, and GeneIII sequences, complimentary forward and reverse oligonucleotides were synthesized by Eurofins Operon. These strands were then annealed together. The oligonucleotides were designed so that the silver fusion prefix and suffix sequences were appended onto the end of each sequence. These parts were then cut with EcoRI and SpeI and ligated into a BioBrick vector. Each of these parts were successfully constructed and sequenced.</p>
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The TorA and HlyA signal peptides were synthesized by DNA 2.0 because these sequences are longer than the other signal peptides, which made the complimentary oligonucleotides method not ideal. The Silver-fusion prefix and suffix was added to each of these constructs. EcoRI and SpeI were used to cut the part out of the commercial vector. The DNA was isolated by gel electrophoresis and ligated into a BioBrick compatible vector, pSB3K3. </p>
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Phasin:</font></b><br>
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The phasin (PhaP) sequence was isolated from the genomic DNA of Cupriavidus necator (also known as Ralstonia eutropha). There are four different phasin genes in the genomic DNA of this organism. This particular phasin was selected based on references in literature, although no information was acquired that indicated that one phasin gene would yield better production over another. The primers were designed so that the Silver-fusion prefix and suffix were overhanging, thereby resulting in a final product that is Silver-fusion compatible. The 579 bp phasin sequence was found to contain a PstI site. The PstI site was mutated using site-directed mutagenesis (LINK TO PROTOCOLS PAGE) (CTGCAG  CTTCAG). Sequencing confirmed that this site was successfully removed.  </p>
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>GFP:</font></b><br>
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Near the beginning of this project, a Silver-fusion compatible GFP BioBrick (BBa_K125500) derived from BBa_E0040 by the Hawaii 2008 iGEM team was obtained. However, upon further analysis it was determined that this part was modified so that the start codon of the sequence was removed. Although this should not affect the expression of GFP in composite parts with a signal peptide prior to the sequence, it is not ideal for this particular project. The lack of a start codon requires N-terminal fusion of another protein or signal peptide, and a functional GFP control without a signal sequence would not be functional. This control is important in our study to compare with composite parts containing signal peptide-protein fusion to determine whether the produced GFP is being transported.  Additionally, this part would not work with C-terminal signal peptide fusions. The HlyA signal peptide is recognized on the C-terminus of the target protein by the Type I secretion pathway (Mergulhão, 2005; Gentschev, 2003; Hess, 1990). The absence of the start codon inhibits study of this secretory pathway. Another disadvantage of this GFP part is its small Stokes shift (excitation 501 nm, emission 511 nm). An ideal GFP that fluorescence would have a shorter excitation wavelength so that GFP-positive samples can be detected visually using a UV transilluminator. </p>
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A new Silver-Fusion compatible GFP BioBrick part was constructed for this project via a similar mechanism as the phasin construct. This particular GFP was previously mutated for improved fluorescence photostability (Crameri, 1996). The excitation and emission wavelengths for this GFP are 395 nm and 501 nm, respectively. That being said, GFP-positive cells emit a bright green fluorescence when exposed to shorter-wavelength UV light, such as on a transilluminator. Primers were synthesized for isolation of the sequence and, like the phasin-specific primers, designed so that the Silver-fusion prefix and suffix were inserted on the ends of the sequence (see primers). Figure X shows GFP- Top10 <i>E. coli</i> colonies (left) and unfused GFP+ Top10 <i>E. coli</i> colonies (right). This figure shows that the GFP construct is functional and easily detectable.</p>
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Figure of GFP goes here<br>
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Bioplastic Production:</font></b><br>
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<p class="class">A plasmid harboring the genes for PHB production (pBHR68) was used in these experiments. This plasmid contains the sequence for ampicillin resistance and contains a ColE1 origin of replication. <i>E. coli</i> harboring pBHR68 were cultured according to methods outlined by Kang et al (2008) and production of PHB was verified using 1H NMR analysis. The spectrum obtained from this experiment is given as Figure X. </p>
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Figure of NMR goes here<br>
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<p class="class">To maintain plasmid compatibility in E. coli transformed with both the pBHR68 and phasin plasmids, it was determined that the vector used for the phasin secretion device required a p15A ori site. BioBrick vector pSB3K3 was found suitable as the host for the secretion constructs. XL1-Blue E. coli were transformed with both a phasin device and the pBHR68 BioBrick plasmids, and these cells were cultured and tested for secretion. </p>
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>SDS-PAGE Analysis</font></b><br>
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<p class="class">Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze the protein content in transformed E. coli. As a positive control, E. coli containing the Lac/RBS/GFP/Terminator (BBa_K208045) construct were sonicated and centrifuged. Additionally, E. coli cells containing an individual BioBrick part (BBa_B0015) were analyzed as a negative control. The resulting gel was stained with coomassie blue and is shown as Figure X. The bright band at 27 kD in the GFP+ sample corresponds to the GFP protein (Bio-Rad). The absence of this band in the GFP- sample further reinforces the functionality of the GFP construct.</p>
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Figure of the GFP gel goes here, could also put glowing tube picture <br>
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<p class="class">The geneIII secretion signal sequence fused to the phasin protein was expressed in E. coli cells. The E. coli cells were grown overnight in LB growth media and centrifuged to pellet the cells.  Supernatants (5ml) were then concentrated using a Centricon Centriplus concentrator (Amicon, Beverly MA).  This process concentrated proteins that were larger than 10kDa and removed molecules smaller than 10kDa.  Approximately 20ug of protein were then applied to a SDS polyacrylamide gel to separate the proteins according to size.  The gel was then stained with coomassie blue for protein detection, as shown in Figure X. Following SDS polyacylamide gel electrophoresis (PAGE) and subsequent coomassie blue staining of the separated proteins, a protein with an approximate size of 22kDA is observed in the sample from the phasin-expressing E. coli cells that is not present in the control E.coli sample.  The phasin protein has been reported by others to migrate on SDS PAGE from 14-28kDa (Pötter, 2002; York, 2002). These results indicate that the GeneIII::phasin expression construct is being produced by the E. coli cells and is being secreted outside the cell into the media.</p>
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Figure of GEneIII gel goes here<br>
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<p class="class">Western blotting with phasin-specific antibodies was performed to verify the observed band as phasin. Figure X shows the apparatus used to transfer proteins onto PVDF paper. Phasin antibody was kindly provided by Anthony J. Sinskey at Massachusetts Institute of Technology. The results of the western blotting were inconclusive. Non-specific binding to larger constructs was observed. Additional testing is required to further reinforce preliminary findings and confirm the secretion of phasin. The secretion of phasin would provide evidence that PHA recovery via phasin secretion is possible. Addtionally, this would reinforce that the constructed BioBricks are not only functional, but would be beneficial for use in other studies.  </p>
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<li>Resolve problems with conversion of broad-host range vectors to BioBrick compatible formats.</li>
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Latest revision as of 02:00, 22 October 2009

USU iGem

Untitled Document

PROJECT Abstract
Introduction
Broad-Host Vectors
Secretion
Experiments
Future Work
References
BioBricks without Borders:
Investigating a multi-host BioBrick vector and secretion of cellular products

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.



Retrieved from "http://2009.igem.org/Team:Utah_State/Project"