Week 10 HW: Imaging and Measurement

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Part 1: Final Project

To validate my “Aragonite Anchor” this semester, I’ll first confirm protein expression and localization using SDS-PAGE and Western Blotting. By fractionating the cells and targeting my specific gene tags, I can prove the OmpA-CBP-1 fusion is actually reaching the outer membrane. To measure the binding affinity to CaCO3, I’ll use Fluorescence Spectroscopy; by tagging the protein with a marker like GFP, I can quantify exactly how much “glue” stays stuck to the crystals after washing.For structural characterization, I’ll use XRD to verify I’ve successfully formed the aragonite phase and SEM to get the “money shot” of the layered, nacre-like morphology. Finally, I’ll test the mechanical strength of the composite via Nanoindentation. This will let me measure the Young’s Modulus and fracture toughness, providing the quantitative data needed to prove my engineered protein actually makes the material stronger than a standard mineral control.

Part 2: Waters HW 1

  1. According to Expasy: Theoretical pI/Mw: 5.90 / 27875.41
  2. To find the molecular weight from the BioAccord spectrum, we select two adjacent peaks. Let’s pick the two most intense peaks: Peak 1: 903.7753 & Peak 2: 933.8860. Using the formula for n that I was given, n ~ 30.98, meaning z ~31. Thus, the peak at 903.7753 has a charge of +31, and the peak at 933.8860 has a charge of +30. Relating m/z, MW and z, I get MW ~ 27,986 Da. To find the mass accuracy, I used the accuracy equation, getting a result of about 3960 ppm. The calculated accuracy of ~3960 ppm is quite high for a high-resolution mass spectrometer like the Waters BioAccord (which typically provides < 20 ppm).A difference of 110.85 Da is very significant. In a synthetic biology context, this suggests the protein being measured is not exactly the theoretical sequence.

Part 3: Waters HW 2

  1. eGFP contains 20 lysines and 7 arginines
  2. 19 peptides will be generated from this digestion
  3. There are about 19 peaks when considered above 10%
  4. Part 3 matches with part 2
  5. The main peak is at m/z 525.76712; this spacing is roughly 0.5, so we can conclude that 2+ is the most abundant charge state. Thus, using equations, we can calculate that the mass is about 1051 Da
  6. The peptide thus most likely be FEGDTLVNR, which has a mass that matches very closely with our calculated mass. This error is roughly 2.8ppm
  7. 88%

Part 4: Water HW 3

To identify the oligomeric species on the CDMS spectrum, we first need to calculate the theoretical masses for each state. The spectrum’s x-axis is in Megadaltons (MDa), so we’ll convert the kilodalton (kDa) subunit masses accordingly.

Based on the subunit masses provided in Table 1:
7FU Subunit: 340 kDa = 0.34 MDa 8FU Subunit: 400 kDa = 0.40 MDa

Thus:

  • 7FU Decamer: 3.40 MDa
  • 8FU Didecamer: 8 MDa
  • 8FU 3-Decamer: 12 MDa
  • 8FU 4-Decamer: 16 MDa

To map this to the CDMS spectrum:
7FU Decamer: This corresponds to the peak labeled 3.4 on the spectrum. It sits just to the left of the larger 4.013 MDa peak.
8FU Didecamer: This corresponds to the very tall, prominent peak labeled 8.33. While the theoretical math suggests 8.0 MDa, the 8.33 peak is the primary didecamer signal in this region for KLH-2. 8FU 3-Decamer: This corresponds to the peak labeled 12.67. This aligns well with the expected 12 MDa range.
8FU 4-Decamer: This corresponds to the small cluster of peaks around the 16-17 MDa mark. While there isn’t a single high-intensity label like the others, the signal intensity between 15 and 20 MDa represents these higher-order oligomers.