Week 10 HW: Advanced Imaging & Measurement Technology

Homework: Final Project

1. Identify at least one aspect of your project that you will measure.

For the DermLogic v2 platform, the primary measurement is the fluorescence intensity generated by the reconstitution of split-sfGFP. This serves as a quantitative proxy for the presence of the target HPV16 L1 and E6 mRNA biomarkers. Additionally, I will measure the molecular weight and structural integrity of the transcribed RNA triggers and the synthesized DNA inserts to ensure the “AND-gate” logic components are correct before assembly.

2. Describe the elements you would like to measure and how you will perform these measurements.

  • Transcriptional Accuracy: I will measure the length and purity of the RNA triggers (L1 and E6) produced via T7 in vitro transcription.
  • Logic Gate Kinetics (AND-gate): I will measure the rate of sfGFP complementation (fluorescence over time). This requires monitoring four conditions: a negative control, each trigger individually, and the dual-trigger state to confirm that signal only occurs in the presence of both.
  • Therapeutic Module Validation: I will measure the expression of the 116 nt antisense E6 RNA segment. Since this is co-transcribed with the reporter, I must verify it is not degraded and maintains its predicted size.
  • Protein Fragment Identity: I need to confirm that the split-sfGFP fragments (N-terminal 1-157 and C-terminal 158-238) are being synthesized correctly within the PURExpress cell-free system.

3. What technologies will you use? Describe in detail.

To validate the Experimental Design of DermLogic v2, I will use:

  • Fluorescence Spectroscopy (Plate Reader): I will use a microplate reader set to Ex 485nm / Em 507nm to perform real-time kinetic assays at 37°C. This allows for the quantification of the sfGFP reconstitution speed and the establishment of the Limit of Detection (LoD).
  • Urea-PAGE (Polyacrylamide Gel Electrophoresis): As specified in Aim 3 of my proposal, I will use Urea-PAGE under denaturing conditions to resolve the RNA transcripts. This is critical for confirming the presence of the antisense/scaffold band and ensuring no premature transcriptional termination.
  • Liquid Chromatography-Mass Spectrometry (LC-MS): Inspired by the Week 10 Lab, I would use Intact Mass Analysis on a system like the Waters Xevo G3 QTof to confirm the molecular weight of the synthesized split-sfGFP fragments.
  • Peptide Mapping (MS/MS): I would use tryptic digestion followed by tandem mass spectrometry to confirm the amino acid sequence of the split-junction (residues 157/158), ensuring the fragments are prepared exactly as designed for optimal reconstitution.