Advanced Imaging & Measurement Technology

Homework: 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.

  • Sanger Sequencing will be utilized to confirm the primary nucleotide sequence of the plasmids. Quantitative Real-Time PCR (qPCR) will be employed to measure mRNA expression levels, ensuring that the genetic instructions are being transcribed at the predicted rates within the cell-free environment.
  • Technology: Cryo-Electron Microscopy (Cryo-EM) will be used to visualize the morphology and assembly of the bacterial microcompartments. Additionally, Dynamic Light Scattering (DLS) will measure the hydrodynamic radius and polydispersity index to ensure a uniform population of nanocages, while Liquid Chromatography-Mass Spectrometry an be used to quantify the ratio of encapsulated versus non-encapsulated proteins.
  • Technology: Fluorimetry and Surface Plasmon Resonance (SPR) will be employed to measure the binding affinity of the activation protein to the circuit’s sensor. By using fluorescent reporter proteins for the therapeutic payload during testing, the “transfer function” or the logic curve of the IANN can be mapped to ensure that the dose-response remains calibrated and autonomous.
  • Technology: Flow Cytometry will be utilized to assess cell viability and the activation state of surrounding immune cells (T cells and NK cells). The LDH (Lactate Dehydrogenase) Release Assay will serve as a primary metric for measuring the killing capacity (cytotoxicity) of the engineered system when exposed to cancer cell lines in vitro.

Homework: Waters Part I — Molecular Weight

  1. Calculated Theoretical Molecular WeightBased on the provided 246 amino acid sequence –> MW: ~27,949.2 Da.
  2. Experimental Molecular Weight Calculation: 27,981.9 Da
  3. Accuracy: 0.117%
  4. z ~= 23

Homework: Waters Part II — Secondary/Tertiary structure

  1. Native vs. Denatured Protein ConformationsThe primary difference between native and denatured protein states in mass spectrometry lies in the surface area and solvent accessibility of the molecule.Native State (Folded): In its natural environment, a protein like eGFP is tightly folded into a compact 3D structure. Most of its basic amino acid residues (like Lysine and Arginine), which accept protons during Electrospray Ionization (ESI), are buried within the protein’s core. Consequently, the protein can only pick up a few charges. This results in:Higher $m/z$ values: Because the charge is low, the mass-to-charge ratio is high.Narrow Distribution: Only a few charge states (peaks) are visible because the folded structure is rigid.Denatured State (Unfolded): When a protein is denatured, it unfolds into a “random coil” or extended string. This exposes nearly all basic sites to the solvent, allowing for significant protonation .Broad Distribution: A wide “envelope” of many charge states is observed because the unfolded protein can adopt various degrees of protonation.

## Homework: Waters Part III

  1. Theoretical Molecular Weight CalculationBased on the provided eGFP sequence (247 amino acids including the LE linker and HHHHHH His-tag):Amino Acid Counts: 20 Lysines (K) and 6 Arginines (R).Average Molecular Weight: Approximately $28,006.3$ Da (unmodified).Matured eGFP (with chromophore): Accounting for the $-20.03$ Da loss during chromophore formation (T66-Y67-G68), the corrected theoretical weight is approximately $27,986.3$ Da.
  2. MW = 27,981.9 Da
  3. 157ppm
  4. Charg =e state ~= 19
  5. Cleavage Sites: 20 Lysines (K) + 6 Arginines (R) = 26 sites.Predicted Peptides: Assuming no missed cleavages, trypsin will generate 27 peptides.Observed Chromatographic Peaks: Between 0.5 and 6 minutes, there are 18 peaks with $>10%$ relative abundance (labeled peaks at 0.43, 0.61, 1.43, 1.80, 1.85, 1.93, 2.17, 2.26, 2.54, 2.78, 3.53, 3.59, 3.70, 4.30, 4.48, 4.64, 4.87, and 5.06).
  6. To identify the peptide eluting at 2.78 minutes, the experimental data from Figure 5b is compared against the theoretical tryptic peptides predicted by the PeptideMass tool. The observed peak has a mass-to-charge ratio of 525.76712. By examining the isotopic spacing in the zoom-in inset, where the difference between peaks is approximately $0.492$ Da, the charge state is determined to be $z = 2$. Using the formula MW = z* (m/z - 1.0078) + 1.0078, the singly charged mass is calculated to be 1050.52644 Da. This matches the theoretical mass of the eGFP tryptic peptide FEGDTLVNR ($1050.514$ Da). The mass accuracy of this measurement is approximately $11.8$ ppm

Homework Part IV:

To find these species, multiply the subunit weight by the number of units and match the total to the labels on the graph’s horizontal axis.

  • 7FU Decamer: This matches the peak labeled 3.4 Megadaltons.

  • 8FU Didecamer: This aligns with the tallest peak on the spectrum, labeled 8.33 Megadaltons.

  • 8FU 3-Decamer: This corresponds to the peak at 12.67 Megadaltons.

  • 8FU 4-Decamer: This is represented by the small cluster of activity visible around the 17 Megadaltons mark.

The reason the experimental peaks (like 8.33) are slightly higher than the calculated weights (like 8.0) is due to “extra” mass from attached sugars or small linker proteins common in these large biological structures.