https://pages.htgaa.org/2026a/ariadna-abigail-ruiz-castro/homework/week-10--advanced-imaging--measurement-technology/index.htmlWeek 10 — Advanced Imaging & Measurement Technology Homework: Final Project For your final project: Please identify at least one (ideally many) aspect(s) of your project that you will measure. It could be the mass or sequence of a protein, the presence, absence, or quantity of a biomarker, etc. Please describe all of the elements you would like to measure, and furthermore describe how you will perform these measurements. What are the technologies you will use (e.g., gel electrophoresis, DNA sequencing, mass spectrometry, etc.)? Describe in detail. Measurement and Validation Techniques for the Bio-Sticker Controlled Gas Exposure Assays The Bio-Sticker will first be tested in sealed exposure chambers containing precisely known concentrations of target toxic gases, such as ammonia or formaldehyde. These chambers allow accurate simulation of hazardous industrial environments while maintaining strict control over temperature, humidity, and gas concentration. By exposing the engineered fungal Bio-Sticker to increasing concentrations of the target analyte, we can determine its activation threshold, sensitivity, and dynamic range. This approach also enables the generation of dose-response curves, which are essential for calibrating the system and defining the concentration at which the color change becomes visible. Colorimetric Analysis The primary readout of the Bio-Sticker is the visible blue color produced by expression of the chromoprotein AmilCP. Colorimetric analysis will be used to quantify this response objectively. Images of the Bio-Sticker will be captured under standardized lighting conditions, and software such as ImageJ will be used to analyze changes in color intensity. Measurements will focus on RGB (red, green, blue) values and, when applicable, absorbance at the wavelength corresponding to AmilCP. This technique allows precise quantification of signal strength, comparison between samples, and monitoring of signal development over time. Digital Image Analysis In addition to simple colorimetric measurements, digital image processing will be employed to evaluate spatial uniformity, signal progression, and long-term stability of the color response. Time-course imaging can be used to track the kinetics of AmilCP expression after exposure to toxic gases. This enables measurement of response time, persistence of the signal, and any degradation or fading over extended periods. Such analyses are particularly important for assessing practical usability in field conditions. Polymerase Chain Reaction (PCR) PCR will be used to confirm successful integration of the engineered genetic circuit into the Aspergillus nidulans genome. Specific primers will be designed to amplify regions spanning the inserted construct and adjacent genomic sequences. Successful amplification of fragments of the expected size will verify the presence of the biosensing cassette. This serves as an initial molecular confirmation that the strain has been correctly engineered. DNA Sequencing Following PCR confirmation, DNA sequencing will be performed to verify the exact nucleotide sequence of the inserted construct. This step ensures that the promoter, sensing elements, reporter gene (AmilCP), and regulatory sequences have been integrated without mutations, deletions, or rearrangements. Sequence verification is critical to ensure that the genetic circuit will function as intended. Reverse Transcription Quantitative PCR (RT-qPCR) RT-qPCR will be used to measure transcriptional activation of the reporter gene after gas exposure. RNA will be extracted from the fungal cells before and after exposure to target gases, converted into complementary DNA (cDNA), and amplified using gene-specific primers. By comparing transcript levels under different conditions, this technique will quantify the extent to which the sensing circuit is activated. RT-qPCR provides highly sensitive, quantitative insight into gene expression dynamics. Spectrophotometry (Optional) Spectrophotometric analysis may be used to complement image-based measurements. Pigments extracted from fungal samples can be analyzed by measuring absorbance at wavelengths specific to AmilCP. This provides an additional quantitative assessment of chromoprotein production and can be particularly useful for validating colorimetric data. Specificity Testing To ensure selectivity, the Bio-Sticker will be exposed not only to target toxic gases but also to non-target compounds commonly present in industrial environments. By comparing responses across these conditions, we can determine whether the system selectively responds to the intended analyte or produces false positives. This is essential for establishing reliability in real-world applications. Stability and Shelf-Life Testing Long-term performance will be evaluated by monitoring the Bio-Sticker under different storage and environmental conditions. Parameters such as baseline color, response capability, and signal durability will be assessed over time. These studies will determine shelf life, operational stability, and robustness under field deployment conditions. Together, these techniques will provide a comprehensive characterization of the Bio-Sticker, from genetic validation to functional performance, ensuring that it operates as a reliable, low-cost, and easily interpretable biosensor for toxic gas detection in hazardous industrial environments. Homework: Waters Part I — Molecular Weight We will analyze an eGFP standard on a Waters Xevo G3 QTof MS system to determine the molecular weight of intact eGFP and observe its charge state distribution in the native and denatured (unfolded) states. The conditions for LC-MS analysis of intact protein cause it to unfold and be detected in its denatured form (due to the solvents and pH used for analysis).Hugoen