Week 10 HW: Advanced Imaging and 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.


Aspect

Measurement Goal

Scientific Rationale

Silk Substrate

Percent weight loss

Quantifies the effectiveness of BmCoc in selectively degrading sericin. 

Bacterial Growth

Colony Forming Units (CFU)

Determines if E. coli proliferates by utilizing degraded sericin as a nutrient source. 

Enzyme Output

Concentration of His-tagged BmCoc

Verifies production, secretion, and stability of the functional enzyme in the supernatant. 




Technologies and Methodologies

1. Gravimetric Analysis (Mass Measurement)

I will perform precise weighing of silk fibers before and after incubation. By using a high-precision analytical balance and drying samples in a desiccator/oven to a constant mass, I will establish a reliable "dry mass" baseline. This is the most direct metric for determining the total degradation of the sericin-fibroin matrix.

2. Microbial Quantification (CFU Assay)

To measure proliferation, I will use serial dilution plating on selective media (LB + Ampicillin). By vortexing the silk fibers with a detergent like Tween20, you detach biofilm-associated cells, allowing for an accurate count of viable bacteria. This confirms that the engineered cells are not just surviving but actively utilizing the silk-derived nutrients.

3. Protein Quantification and Verification

  • Bradford Assay: This colorimetric assay will be used for the rapid, high-throughput quantification of the total His-tagged BmCoc protein recovered from the culture supernatant. It provides a standard estimate of the enzyme titer.

  • Ni-NTA Affinity Chromatography: Before quantification, I will use Ni-NTA resin to specifically capture the His-tagged BmCoc. This serves as a vital purification step, removing background E. coli proteins and allowing for a more accurate concentration measurement of your enzyme of interest.

  • A280 Absorbance: Using a NanoDrop spectrophotometer, I will perform direct UV absorption measurements at 280 nm, utilizing the calculated extinction coefficient of BmCoc to provide a secondary confirmation of the protein concentration.

Homework: Waters Part I — Molecular Weight

  1. Based on the predicted amino acid sequence of eGFP (see below) and any known modifications, what is the calculated molecular weight? You can use an online calculator like the one at https://web.expasy.org/compute_pi/
    eGFP Sequence:
    MVSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKQ HDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YIMADKQKNG IKVNFKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT LGMDELYKLE HHHHHH
    Note: This contains a His-purification tag (HHHHHH) and a linker (the LE before it).

Theoretical pI/Mw: 5.90 / 28006.60

  1. Calculate the molecular weight of the eGFP using the adjacent charge state approach described in the recitation. Select two charge states from the intact LC-MS data (Figure 1) and:

    1. Determine  for each adjacent pair of peaks  using:

n = 875,4421

n+1 = 903,7148

z = -31,9638

2. Mw = (m/z * z) = (875,4421*-31.9638) = -27.982,4562 Da

3. Accuracy = (MWexperiment - MWtheory) / MWtheory 

         = (27982,4562 - 28006,60) / 28006,60 

= -0,0008 

   3. Can you observe the charge state for the zoomed-in peak in the mass spectrum for the intact eGFP? If yes, what is it? If no, why not?

You cannot observe the charge state of the zoomed-in peak in the mass spectrum for the intact eGFP, as the protein is unfolded and in its denatured state. Therefore the protein is in a larger charged state, thus it can not be observed.    


Homework: Waters Part III — Peptide Mapping - primary structure

  1. How many Lysines (K) and Arginines (R) are in eGFP? Please circle or highlight them in the eGFP sequence given in Waters Part I question 1 above. (Note: adding the sequence to Benchling as an amino acid file and clicking biochemical properties tab will show you a count for each amino acid).

MVSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKQ HDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YIMADKQKNG IKVNFKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT LGMDELYKLE HHHHHH  
There are 20 Lysines and 6 Arganines in eGFP

How many peptides will be generated from tryptic digestion of eGFP?

Based on the Expasy calculator, 21 peptides generated when using trypsin to perform the digest.

Based on the LC-MS data for the Peptide Map data generated in lab (please use Figure 5a as a reference) how many chromatographic peaks do you see in the eGFP peptide map between 0.5 and 6 minutes? You may count all peaks that are >10% relative abundance.

I count 21 peaks in the generated peptide map. 

Assuming all the peaks are peptides, does the number of peaks match the number of peptides predicted from question 2 above? Are there more peaks in the chromatogram or fewer?

When comparing the generated peptides from the expasy calculator and the LC-MS data for the Peptide Map data generated in lab, they both indicate that 21 peptides have been created.

Identify the mass-to-charge (m/z) of the peptide shown in Figure 5b. What is the charge (z) of the most abundant charge state of the peptide (use the separation of the isotopes to determine the charge state). Calculate the mass of the singly charged form of the peptide (M+H^+) based on its m/z and z.

n =521.7612

n+1 = 526.25918


z = (m/zn+1) / ( (m/zn) – (m/zn+1) )

   = 526.25918 / (521.7612 – 526.25918)

   = 526.25918 /  -4,49798

   = -116,999004 = 117

m =  m / z  * z 

    =  526.25918 / 117

    = 4,49794171

What is the percentage of the sequence that is confirmed by peptide mapping? (see Figure 6)

The percentage of the sequence that is confirmed by peptide mapping is 88%


Homework: Waters Part IV — Did I make GFP?


We will determine Keyhole Limpet Hemocyanin (KLH)’s oligomeric states using charge detection mass spectrometry (CDMS). CDMS single-particle measurements of KLH allow us to make direct mass measurements to determine what oligomeric states (that is, how many protein subunits combine) are present in solution. Using the known masses of the polypeptide subunits (Table 1) for KLH, identify where the following oligomeric species are on the spectrum shown below from the CDMS (Figure 7):

  • 7FU Decamer = 10 subunits  340 * 10 = 3400 kDa = 3.4 MDa ≈

  • 8FU Didecamer = 20 subunits  400 * 20 = 8000 kDa = 8.0 MDa  ≈ 8.33 MDa

  • 8FU 3-Decamer = 30 subunits 400 * 30 = 1200 kDA = 12.0 MDa ≈ 12.67 MDA

  • 8FU 4-Decamer = 40 subunits 400 * 40 = 1600 kDA = 16.0 MDa 

The calculated weights somewhat correspond to peaks in the CDMS chart, taking possible extraneous digits into consideration when looking at the provided data.