Rapid optimization of gene dosage in E. coli using DIAL strains

<p>Abstract</p> <p>Background</p> <p>Engineers frequently vary design parameters to optimize the behaviour of a system. However, synthetic biologists lack the tools to rapidly explore a critical design parameter, gene expression level, and have no means of systematically varying the dosage of an entire genetic circuit. As a step toward overcoming this shortfall, we have developed a technology that enables the same plasmid to be maintained at different copy numbers in a set of closely related cells. This provides a rapid method for exploring gene or cassette dosage effects.</p> <p>Results</p> <p>We engineered two sets of strains to constitutively provide a <it>trans</it>-acting replication factor, either Pi of the R6K plasmid or RepA of the ColE2 plasmid, at different doses. Each DIAL (different allele) strain supports the replication of a corresponding plasmid at a constant level between 1 and 250 copies per cell. The plasmids exhibit cell-to-cell variability comparable to other popular replicons, but with improved stability. Since the origins are orthogonal, both replication factors can be incorporated into the same cell. We demonstrate the utility of these strains by rapidly assessing the optimal expression level of a model biosynthetic pathway for violecein.</p> <p>Conclusions</p> <p>The DIAL strains can rapidly optimize single gene expression levels, help balance expression of functionally coupled genetic elements, improve investigation of gene and circuit dosage effects, and enable faster development of metabolic pathways.</p

Eugene – A Domain Specific Language for Specifying and Constraining Synthetic Biological Parts, Devices, and Systems

BACKGROUND: Synthetic biological systems are currently created by an ad-hoc, iterative process of specification, design, and assembly. These systems would greatly benefit from a more formalized and rigorous specification of the desired system components as well as constraints on their composition. Therefore, the creation of robust and efficient design flows and tools is imperative. We present a human readable language (Eugene) that allows for the specification of synthetic biological designs based on biological parts, as well as provides a very expressive constraint system to drive the automatic creation of composite Parts (Devices) from a collection of individual Parts. RESULTS: We illustrate Eugene's capabilities in three different areas: Device specification, design space exploration, and assembly and simulation integration. These results highlight Eugene's ability to create combinatorial design spaces and prune these spaces for simulation or physical assembly. Eugene creates functional designs quickly and cost-effectively. CONCLUSIONS: Eugene is intended for forward engineering of DNA-based devices, and through its data types and execution semantics, reflects the desired abstraction hierarchy in synthetic biology. Eugene provides a powerful constraint system which can be used to drive the creation of new devices at runtime. It accomplishes all of this while being part of a larger tool chain which includes support for design, simulation, and physical device assembly

Development of Point-of-Care Diagnostic Technologies Utilizing Aqueous Two-Phase Systems

Infectious diseases are one of the major causes of death in developing countries. These diseases are caused by pathogenic organisms, such as bacteria, viruses, and parasites. Current gold standard methods of detection include cell culturing, the enzyme-linked immunosorbent assay (ELISA), and the polymerase chain reaction (PCR); however, these methods are often complex, have a long time-to-result, and require expensive equipment and trained personnel. Such limitations make it difficult for these standard diagnostics to be used in resource-poor settings. Unfortunately, it is also these developing countries that could currently benefit most from these early diagnosis assays. Therefore, there is a growing need for simple, sensitive, and efficient diagnostic methods. To this end, researchers have made efforts to design diagnostics with the aim to be viable at the point-of-care (POC). While there have been great advances in converting complicated laboratory-based assays into POC-friendly diagnostics, the ability to simplify the method while maintaining the diagnostic test’s effectiveness remains a primary concern. Often, low assay sensitivity as a result of poor processing of samples in complex media or low concentration of biomarkers are the main challenges.One example of a POC-friendly diagnostic is the paper-based lateral-flow immunoassay (LFA). While the advantages of the LFA are that it is low-cost, rapid, user-friendly, and does not require laboratory equipment, the main drawback of the LFA is that it is not as sensitive as traditional laboratory tests. To address this problem, our laboratory has previously utilized aqueous two-phase systems (ATPSs) to concentrate biomarkers via partitioning into one of the two phases of an ATPS prior to its application to the LFA. Using this pre-concentration step, the detection limit of the LFA was improved 10-fold.While our lab has had much success in combining ATPSs and LFA to predictably concentrate biomarkers and improve the LFA limit of detection, this thesis expands the application of ATPSs for the development of other POC diagnostic formats. Chapter 2 describes the application of an ATPS to a paper-based spot immunoassay for detection of foodborne pathogens in food samples. We designed a spot immunoassay that utilizes a UCON-potassium phosphate salt ATPS for the pre-concentration of Escherichia coli (E. coli) O157:H7. This platform was tested with samples of O157:H7 spiked in phosphate-buffered saline (PBS) and milk. The ATPS was found to improve the detection limit of the spot test, yielding detection in milk at 106 colony forming units (cfu)/mL within 30 min. In Chapter 3, we extended the application of ATPSs to nucleic acid amplification tests (NAATs) by integrating an ATPS with isothermal DNA amplification. We introduced a novel system that combines thermophilic helicase-dependent amplification (tHDA) with a Triton X-100 micellar ATPS to achieve cell lysis, lysate processing, and enhanced nucleic acid amplification in a simple, one-step process. The combined one-pot system was able to detect whole cell samples containing as few as 102 cfu/mL of E. coli, making it competitive to existing gold standard NAATs. Moreover, the one-pot reaction improved the detection limit of tHDA by 105-fold, and is the first known application of ATPSs to isothermal DNA amplification. This significant improvement in the detection limit was attributed to the synergistic effects of DNA purification and concentration in the ATPS, which rendered the one-pot reaction much more effective at processing whole cell samples compared to the conventional tHDA reaction. While we successfully tested our one-pot system with E. coli as a model pathogen, our system’s ease-of-use, sensitivity, and tunability underline its potential as a POC diagnostic platform to detect for a variety of infectious diseases. After demonstrating success with our one-pot reaction, we addressed two challenges that would help further drive the development of a POC NAAT. Specifically, these corresponded to the limited understanding of how to use an ATPS as a sample preparation method and the need to use liquid, test tube-based reactions for the current NAAT technology that could cause difficulties in storage and transportation for POC applications. In Chapter 4, we addressed these challenges by first developing a mathematical model for DNA partitioning to determine which design parameters should be considered for optimal nucleic acid partitioning in a chosen ATPS. Secondly, we assembled a device to perform Recombinase Polymerase Amplification (RPA) and designed an LFA to subsequently detect the amplicons on paper. After development of our model, we identified the electrostatic potential difference and the size of the DNA as potential factors that could influence DNA partitioning. Using these parameters, we determined that a Triton X-114 ATPS containing Mg(CH3COO)2 salt should be used to ensure greater partitioning into the micelle-poor phase. After verifying that our system was optimal for partitioning large genomic DNA fragments, we applied this ATPS as a genomic DNA sample pre-concentration step for the improvement of RPA. Not only did we successfully design and perform RPA on a paper matrix, but we also achieved a 10-fold improvement in the detection limit when our ATPS DNA pre-concentration method was combined with paper-based RPA and LFA. Ultimately, we hope that this increased understanding of DNA partitioning behavior in ATPSs and application of NAAT steps to paper-based formats can lead to better engineered designs to further advance the NAAT for POC use

Development of Point-of-Care Diagnostic Technologies Utilizing Aqueous Two-Phase Systems

Infectious diseases are one of the major causes of death in developing countries. These diseases are caused by pathogenic organisms, such as bacteria, viruses, and parasites. Current gold standard methods of detection include cell culturing, the enzyme-linked immunosorbent assay (ELISA), and the polymerase chain reaction (PCR); however, these methods are often complex, have a long time-to-result, and require expensive equipment and trained personnel. Such limitations make it difficult for these standard diagnostics to be used in resource-poor settings. Unfortunately, it is also these developing countries that could currently benefit most from these early diagnosis assays. Therefore, there is a growing need for simple, sensitive, and efficient diagnostic methods. To this end, researchers have made efforts to design diagnostics with the aim to be viable at the point-of-care (POC). While there have been great advances in converting complicated laboratory-based assays into POC-friendly diagnostics, the ability to simplify the method while maintaining the diagnostic test’s effectiveness remains a primary concern. Often, low assay sensitivity as a result of poor processing of samples in complex media or low concentration of biomarkers are the main challenges.One example of a POC-friendly diagnostic is the paper-based lateral-flow immunoassay (LFA). While the advantages of the LFA are that it is low-cost, rapid, user-friendly, and does not require laboratory equipment, the main drawback of the LFA is that it is not as sensitive as traditional laboratory tests. To address this problem, our laboratory has previously utilized aqueous two-phase systems (ATPSs) to concentrate biomarkers via partitioning into one of the two phases of an ATPS prior to its application to the LFA. Using this pre-concentration step, the detection limit of the LFA was improved 10-fold.While our lab has had much success in combining ATPSs and LFA to predictably concentrate biomarkers and improve the LFA limit of detection, this thesis expands the application of ATPSs for the development of other POC diagnostic formats. Chapter 2 describes the application of an ATPS to a paper-based spot immunoassay for detection of foodborne pathogens in food samples. We designed a spot immunoassay that utilizes a UCON-potassium phosphate salt ATPS for the pre-concentration of Escherichia coli (E. coli) O157:H7. This platform was tested with samples of O157:H7 spiked in phosphate-buffered saline (PBS) and milk. The ATPS was found to improve the detection limit of the spot test, yielding detection in milk at 106 colony forming units (cfu)/mL within 30 min. In Chapter 3, we extended the application of ATPSs to nucleic acid amplification tests (NAATs) by integrating an ATPS with isothermal DNA amplification. We introduced a novel system that combines thermophilic helicase-dependent amplification (tHDA) with a Triton X-100 micellar ATPS to achieve cell lysis, lysate processing, and enhanced nucleic acid amplification in a simple, one-step process. The combined one-pot system was able to detect whole cell samples containing as few as 102 cfu/mL of E. coli, making it competitive to existing gold standard NAATs. Moreover, the one-pot reaction improved the detection limit of tHDA by 105-fold, and is the first known application of ATPSs to isothermal DNA amplification. This significant improvement in the detection limit was attributed to the synergistic effects of DNA purification and concentration in the ATPS, which rendered the one-pot reaction much more effective at processing whole cell samples compared to the conventional tHDA reaction. While we successfully tested our one-pot system with E. coli as a model pathogen, our system’s ease-of-use, sensitivity, and tunability underline its potential as a POC diagnostic platform to detect for a variety of infectious diseases. After demonstrating success with our one-pot reaction, we addressed two challenges that would help further drive the development of a POC NAAT. Specifically, these corresponded to the limited understanding of how to use an ATPS as a sample preparation method and the need to use liquid, test tube-based reactions for the current NAAT technology that could cause difficulties in storage and transportation for POC applications. In Chapter 4, we addressed these challenges by first developing a mathematical model for DNA partitioning to determine which design parameters should be considered for optimal nucleic acid partitioning in a chosen ATPS. Secondly, we assembled a device to perform Recombinase Polymerase Amplification (RPA) and designed an LFA to subsequently detect the amplicons on paper. After development of our model, we identified the electrostatic potential difference and the size of the DNA as potential factors that could influence DNA partitioning. Using these parameters, we determined that a Triton X-114 ATPS containing Mg(CH3COO)2 salt should be used to ensure greater partitioning into the micelle-poor phase. After verifying that our system was optimal for partitioning large genomic DNA fragments, we applied this ATPS as a genomic DNA sample pre-concentration step for the improvement of RPA. Not only did we successfully design and perform RPA on a paper matrix, but we also achieved a 10-fold improvement in the detection limit when our ATPS DNA pre-concentration method was combined with paper-based RPA and LFA. Ultimately, we hope that this increased understanding of DNA partitioning behavior in ATPSs and application of NAAT steps to paper-based formats can lead to better engineered designs to further advance the NAAT for POC use