Week 10 HW: Advanced Imaging & Measurement Technology

Final Project

Aspects of the project that will be measured

Several aspects of the nanoparticle-mediated plant transfection system will be measured to evaluate delivery efficiency, functional gene expression, and electrophysiological response.

The primary measurable parameters include:

• efficiency of nanoparticle-mediated DNA delivery into plant tissues
• expression of fluorescent reporter proteins
• functional activity of Channelrhodopsin-2
• electrophysiological responses after light stimulation
• physicochemical properties of peptide-based nanoparticles (size and charge)

Additional measurements may include comparison between magnetic nanoparticles and peptide-based nanoparticles in terms of transfection efficiency and signal intensity.

Elements to be measured and methods of measurement

Nanoparticle formation and stability

The size and surface charge of peptide–DNA nanoparticles will be evaluated to confirm successful self-assembly and stability.

Measurements:

• nanoparticle diameter
• zeta potential
• aggregation behavior

Methods:

• dynamic light scattering (DLS)
• zeta potential analysis (under consideration)
• microscopy-based visualization

Presence of plasmid DNA

Successful incorporation of plasmid DNA into nanoparticles will be verified.

Measurements:

• presence and integrity of plasmid DNA
• DNA binding efficiency.

Methods:

• agarose gel electrophoresis
• PCR
• gel retardation assay

Gene expression in plant tissues

Successful transfection will be assessed through expression of GFP reporter.

Measurements:

• fluorescence intensity
• localization of expression

Methods:

• fluorescence microscopy
• confocal microscopy (if available)
• image analysis software

Functional activity of Channelrhodopsin-2

The activity of the optogenetic ion channel will be evaluated after blue-light stimulation.

Measurements:

• changes in membrane potential
• electrophysiological response amplitude
• signal timing after stimulation

Methods:

• extracellular electrode recording
• optical stimulation using blue LED light

Technologies used

1. Fluorescence Microscopy Fluorescence microscopy will be used to detect expression of reporter proteins such as GFP or mCherry in plant tissues. This provides visual confirmation of successful transfection and allows comparison of delivery efficiency between nanoparticle systems.

2. Dynamic Light Scattering (DLS) DLS will be used to characterize nanoparticle size distribution and stability in solution. Nanoparticle size is an important parameter influencing plant tissue penetration and delivery efficiency.

3. Electrophysiological Recording Extracellular electrophysiological measurements will be used to detect electrical responses generated after activation of Channelrhodopsin-2 by blue light stimulation. These measurements will provide functional validation of successful gene expression.

4. Agarose Gel Electrophoresis Agarose gel electrophoresis will be used to verify plasmid DNA integrity and evaluate peptide–DNA complex formation. Gel retardation assays can demonstrate successful nanoparticle assembly by reduced migration of DNA complexes through the gel matrix.

5. Computational Design Tools Benchling will be used for plasmid design, sequence annotation, and in silico cloning simulations. Automation workflows for nanoparticle preparation may additionally be developed using Python scripting and Opentrons OT-2 protocols.

Waters Part I — Molecular Weight

1. His-tagged and linker-containing eGFP MWth = 28,006.60Da, according to ExPASy portal calculator

2. I chose the following peaks: m/z_n = 1000.4302 m/z_n+1 = 965.9684

Using the first formula: z = (m/z_n+1) / (m/z_n - m/z_n+1) z = 965.9684 / (1000.4302 - 965.9684) z = 28.03 meaning the higher m/z peak is 28 and the lower is 29

Calculating molecular weight: MW = z × (m/z - proton mass) for the higher peak, MW = 28 × (1000.4302 - 1.0073) MW = 27,983.84 Da for the lower peak, MW = 29 × (965.9684 - 1.0073) MW = 27,983.87 Da Thus, average experimental MW MW_exp = (27,983.84 + 27,983.87) / 2 MW_exp = 27,983.86 Da

Calculating mass accuracy against the mature theoretical MW: MW_theory = 27,986.57 Da MW_exp = 27,983.86 Da

Accuracy = |MW_exp - MW_theory| / MW_theory Accuracy = |27,983.86 - 27,986.57| / 27,986.57 Accuracy = 0.00009699 = 0.0097% = 97 ppm

3. In the zoomed-in peak around m/z ≈ 1473. If moving toward higher m/z and decreasing the charge by one for each peak, charge state can be estimated from isotope spacing. Δm/z ≈ 0.053 hence, z = 1 / 0.053 z ≈ 19 Thus, we can observe the charge state for the zoomed-in peak in the intact eGFP mass spectrum.

Waters Part III — Peptide Mapping - primary structure

1. In the given sequence,

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKLEHHHHHH

Lysine (K) = 20 Arginine (R) = 6 Total K/R cleavage-related residues = 26

2. ExPASy PeptideMass tool with the parameters given in Figure 4 outputs 19 peptides.

3. The eGFP peptide map (Figure 5a) contains 21 chromatographic peaks between 0.5 and 6 minutes with >10% relative abundance. These are the labeled peaks at retention times (min):

• 0.61
• 0.79
• 1.20
• 1.43
• 1.80
• 1.85
• 1.93
• 2.17
• 2.26
• 2.54
• 2.78
• 3.27
• 3.53
• 3.59
• 3.70
• 4.30
• 4.48
• 4.64
• 4.87
• 5.06
• 5.43

All of them exceed 10% relative abundance.

4. No, they differ. There are slightly more observed chromatographic peaks than predicted peptides (21 vs. 19).

• Some peptides may co-elute at the same retention time
• Some peptides may be too low in abundance to detect
• Some peptides may ionize poorly
• Some peaks may include modified peptides, missed-cleavage products, or non-peptide signals
• The chromatogram peak count is not always equal to the theoretical peptide count

5. The most abundant peak in the spectrum is at m/z 525.76712. The isotope spacing is approximately 0.49206 ≈ 0.5 m/z isotope spacing is approximately: 1 / z => 2 Thus, the most abundant peptide is 2+

[M+H]+ = 2 × 525.76712 - 1 × 1.007276 [M+H]+ = 1050.53 Da

6. By comparing determined [M+H]+ ≈ 1050.53 Da with the theoretical peptide masses from the ExPASy PeptideMass tool, the peptide eluting at 2.78 min most likely corresponds to: FEGDTLVNR. The theoretical monoisotopic mass ([M+H]+) is: 1050.5214 Da ppm = (Δ / theoretical mass) × 1,000,000 ppm ≈ 5.25 ppm

7. From Figure 6, 88% of the eGFP sequence is confirmed by peptide mapping

8. Fragment masses (122.07, 214.09, 388.22, 501.31, 602.35, 537.25, 774.41, 903.44, 1050.52) match the b- and y-ion series for FEGDTLVNR. Key y-ions: y1 (R) = 175.12, y2 (NR) = 289.16, y7 (GDTLVNR), and the immonium ion at 122.07 corresponds to F (phenylalanine immonium). Confirms FEGDTLVNR

Waters Part IV — Oligomers

The 4.013 MDa peak is likely an 8FU decamer (10 × 400 = 4.0 MDa). The 0.1982, 0.79, 1.52 peaks are sub-decameric assemblies/free subunits.

Waters Part V — Did I Make GFP?

ppm error = |28,007 - 28,097| / 28,097 × 1,000,000 ppm error ≈ 3,203 ppm Combined with 88% peptide map coverage and confirmed FEGDTLVNR fragmentation → yes, this is eGFP