Making More Sensitive DNA Sensors Using Gold Nanoparticles and DNA-Based Amplification Networks

This study focuses on the use of gold nanoparticles with DNA-based signal amplification as a detection method for low concentrations of DNA bio-markers. Biotechnology is a rapidly evolving field with primarily medical applications. Early detection is a challenging process for some cancers and other diseases yet is so critical to successful treatment. Increasingly sensitive detection techniques are being developed, but the current gold standard for detecting nucleotide biomarkers at low concentrations is polymerase chain reaction. While this technique is sensitive, it requires the use of active enzymes, a thermocycler, and trained personnel working in a clean environment, and is thus not very feasible for diagnosing diseases in remote locations or third world epidemic scenarios. Gold nanoparticles with complimentary DNA probes provide an easy, colorimetric method for detecting a DNA target, but are not very sensitive to sub-nanomolar concentrations without post-hybridization enhancement or sensitive instruments. To overcome this limitation, we employ enzyme-free, DNA-based amplification networks that use cascading hybridization reactions to produce multiple nanoparticle binding events per molecule of target DNA. Our data show that the DNA-based amplification does increase sensitivity of our colorimetric gold nanoparticles without sacrificing their ease of use. We also expand this detection method to other biomolecule of interest, by using an aptamer sequence to bind a small biomolecule and then trigger the DNA-based amplification network

A synthetic biosensor to detect peroxisomal acetyl-CoA concentration for compartmentalized metabolic engineering

Background Sub-cellular compartmentalization is used by cells to create favorable microenvironments for various metabolic reactions. These compartments concentrate enzymes, separate competing metabolic reactions, and isolate toxic intermediates. Such advantages have been recently harnessed by metabolic engineers to improve the production of various high-value chemicals via compartmentalized metabolic engineering. However, measuring sub-cellular concentrations of key metabolites represents a grand challenge for compartmentalized metabolic engineering. Methods To this end, we developed a synthetic biosensor to measure a key metabolite, acetyl-CoA, in a representative compartment of yeast, the peroxisome. This synthetic biosensor uses enzyme re-localization via PTS1 signal peptides to construct a metabolic pathway in the peroxisome which converts acetyl-CoA to polyhydroxybutyrate (PHB) via three enzymes. The PHB is then quantified by HPLC. Results The biosensor demonstrated the difference in relative peroxisomal acetyl-CoA availability under various culture conditions and was also applied to screening a library of single knockout yeast mutants. The screening identified several mutants with drastically reduced peroxisomal acetyl-CoA and one with potentially increased levels. We expect our synthetic biosensors can be widely used to investigate sub-cellular metabolism and facilitate the “design-build-test” cycle of compartmentalized metabolic engineering

Enhanced DNA Sensing via Catalytic Aggregation of Gold Nanoparticles

A catalytic colorimetric detection scheme that incorporates a DNA-based hybridization chain reaction into gold nanoparticles was designed and tested. While direct aggregation forms an inter-particle linkagefrom only one target DNA strand, catalytic aggregation forms multiple linkages from a single target DNA strand. Gold nanoparticles were functionalized with thiol-modified DNA strands capable of undergoing hybridization chain reactions. The changes in their absorption spectra were measured at different times and target concentrations and compared against direct aggregation. Catalytic aggregation showed a multifold increase in sensitivity at low target concentrations when compared to direct aggregation. Gelelectrophoresis was performed to compare DNA hybridization reactions in catalytic and direct aggregation schemes, and the product formation was confirmed in the catalytic aggregation scheme at low levels of target concentrations. The catalytic aggregation scheme also showed high target specificity. This application of a DNA reaction network to gold nanoparticle-based colorimetric detection enables highly-sensitive, field-deployable, colorimetric readout systems capable of detecting a variety of biomolecules

Targeted mutagenesis and high-throughput screening of diversified gene and promoter libraries for isolating gain-of-function mutations

Targeted mutagenesis of a promoter or gene is essential for attaining new functions in microbial and protein engineering efforts. In the burgeoning field of synthetic biology, heterologous genes are expressed in new host organisms. Similarly, natural or designed proteins are mutagenized at targeted positions and screened for gain-of-function mutations. Here, we describe methods to attain complete randomization or controlled mutations in promoters or genes. Combinatorial libraries of one hundred thousands to tens of millions of variants can be created using commercially synthesized oligonucleotides, simply by performing two rounds of polymerase chain reactions. With a suitably engineered reporter in a whole cell, these libraries can be screened rapidly by performing fluorescence-activated cell sorting (FACS). Within a few rounds of positive and negative sorting based on the response from the reporter, the library can rapidly converge to a few optimal or extremely rare variants with desired phenotypes. Library construction, transformation and sequence verification takes 6–9 days and requires only basic molecular biology lab experience. Screening the library by FACS takes 3–5 days and requires training for the specific cytometer used. Further steps after sorting, including colony picking, sequencing, verification, and characterization of individual clones may take longer, depending on number of clones and required experiments

Raman chemometric urinalysis (Rametrix) as a screen for bladder cancer.

Bladder cancer (BCA) is relatively common and potentially recurrent/progressive disease. It is also costly to detect, treat, and control. Definitive diagnosis is made by examination of urine sediment, imaging, direct visualization (cystoscopy), and invasive biopsy of suspect bladder lesions. There are currently no widely-used BCA-specific biomarker urine screening tests for early BCA or for following patients during/after therapy. Urine metabolomic screening for biomarkers is costly and generally unavailable for clinical use. In response, we developed Raman spectroscopy-based chemometric urinalysis (Rametrix™) as a direct liquid urine screening method for detecting complex molecular signatures in urine associated with BCA and other genitourinary tract pathologies. In particular, the RametrixTM screen used principal components (PCs) of urine Raman spectra to build discriminant analysis models that indicate the presence/absence of disease. The number of PCs included was varied, and all models were cross-validated by leave-one-out analysis. In Study 1 reported here, we tested the Rametrix™ screen using urine specimens from 56 consented patients from a urology clinic. This proof-of-concept study contained 17 urine specimens with active BCA (BCA-positive), 32 urine specimens from patients with other genitourinary tract pathologies, seven specimens from healthy patients, and the urinalysis control SurineTM. Using a model built with 22 PCs, BCA was detected with 80.4% accuracy, 82.4% sensitivity, 79.5% specificity, 63.6% positive predictive value (PPV), and 91.2% negative predictive value (NPV). Based on the number of PCs included, we found the RametrixTM screen could be fine-tuned for either high sensitivity or specificity. In other studies reported here, RametrixTM was also able to differentiate between urine specimens from patients with BCA and other genitourinary pathologies and those obtained from patients with end-stage kidney disease (ESKD). While larger studies are needed to improve RametrixTM models and demonstrate clinical relevance, this study demonstrates the ability of the RametrixTM screen to differentiate urine of BCA-positive patients. Molecular signature variances in the urine metabolome of BCA patients included changes in: phosphatidylinositol, nucleic acids, protein (particularly collagen), aromatic amino acids, and carotenoids

Table1_Targeted mutagenesis and high-throughput screening of diversified gene and promoter libraries for isolating gain-of-function mutations.docx

Targeted mutagenesis of a promoter or gene is essential for attaining new functions in microbial and protein engineering efforts. In the burgeoning field of synthetic biology, heterologous genes are expressed in new host organisms. Similarly, natural or designed proteins are mutagenized at targeted positions and screened for gain-of-function mutations. Here, we describe methods to attain complete randomization or controlled mutations in promoters or genes. Combinatorial libraries of one hundred thousands to tens of millions of variants can be created using commercially synthesized oligonucleotides, simply by performing two rounds of polymerase chain reactions. With a suitably engineered reporter in a whole cell, these libraries can be screened rapidly by performing fluorescence-activated cell sorting (FACS). Within a few rounds of positive and negative sorting based on the response from the reporter, the library can rapidly converge to a few optimal or extremely rare variants with desired phenotypes. Library construction, transformation and sequence verification takes 6–9 days and requires only basic molecular biology lab experience. Screening the library by FACS takes 3–5 days and requires training for the specific cytometer used. Further steps after sorting, including colony picking, sequencing, verification, and characterization of individual clones may take longer, depending on number of clones and required experiments.</p

Table2_Targeted mutagenesis and high-throughput screening of diversified gene and promoter libraries for isolating gain-of-function mutations.docx

Targeted mutagenesis of a promoter or gene is essential for attaining new functions in microbial and protein engineering efforts. In the burgeoning field of synthetic biology, heterologous genes are expressed in new host organisms. Similarly, natural or designed proteins are mutagenized at targeted positions and screened for gain-of-function mutations. Here, we describe methods to attain complete randomization or controlled mutations in promoters or genes. Combinatorial libraries of one hundred thousands to tens of millions of variants can be created using commercially synthesized oligonucleotides, simply by performing two rounds of polymerase chain reactions. With a suitably engineered reporter in a whole cell, these libraries can be screened rapidly by performing fluorescence-activated cell sorting (FACS). Within a few rounds of positive and negative sorting based on the response from the reporter, the library can rapidly converge to a few optimal or extremely rare variants with desired phenotypes. Library construction, transformation and sequence verification takes 6–9 days and requires only basic molecular biology lab experience. Screening the library by FACS takes 3–5 days and requires training for the specific cytometer used. Further steps after sorting, including colony picking, sequencing, verification, and characterization of individual clones may take longer, depending on number of clones and required experiments.</p