Week 10 HW: Imaging and measurement

Homework: Final Project

1. Conjugation Frequency (Quantitative HGT Efficiency)

What is measured: The ratio of transconjugants to total recipient cells — the primary numerical output of the experiment. After overnight incubation on LB agar, cells are re-printed onto:

• Plate A (no antibiotic) → counts all recipient colonies (pink) + donor (blue)
• Plate B (+ ampicillin 100 µg/mL) → only AmpR transconjugants survive in recipient zones

Conjugation frequency = N(blue AmpR in recipient zone) / N(total recipient on Plate A)

Technologies: Automated colony counting via high-resolution flatbed scanning + ImageJ / OpenCFU software

2. Chromogenic Phenotype — Colorimetric Measurement (pink → blue transition)

What is measured: The spatial and quantitative shift in colony colour as a direct proxy for bglA expression and β-glucosidase enzymatic activity in transconjugants.

• Flatbed scan of agar plates at high resolution (≥600 dpi)
• Extract RGB pixel values per colony zone using ImageJ or Python
• Pink colonies: H ≈ 330–350°; blue transconjugants: H ≈ 170–190°
• Quantify the fractional area of blue signal within recipient-zone boundaries

Technologies: Digital image analysis (ImageJ, ColourDeconvolution plugin)

3. Plasmid Presence in Transconjugants — Colony PCR

What is measured: Physical confirmation that the mobilizable plasmid (carrying oriT_RK2–Ptrc–RBS–bglA) has transferred into recipient cells.

• Pick 12–24 blue AmpR colonies from Plate B recipient zones
• Colony PCR with primers flanking bglA CDS (~950 bp amplicon) and a second primer pair spanning the oriT–Ptrc junction
• Controls: donor lysate (positive), untransformed recipient (negative), water (NTC)

Technologies:

• ATC Thermal Cycler using 96-Armadillo PCR plates
• Reaction setup: Echo525 acoustic liquid handler for master mix dispensing into 384-well format (high-throughput screening of many colonies)
• 1% agarose gel electrophoresis with ethidium bromide / SYBR Safe staining, UV transilluminator; band at ~950 bp confirms transfer

4. β-Glucosidase Enzymatic Activity — pNPG Colorimetric Assay

What is measured: Direct enzymatic activity of the bglA gene product (aryl-phospho-β-D-glucosidase from E. faecalis) in cell lysates — the most quantitative functional readout.

• Grow 3–5 blue transconjugant colonies overnight → pellet → lyse by bead-beating or freeze-thaw
• Add pNPG (para-nitrophenyl-β-D-glucopyranoside) substrate to lysate in 96-well microplate
• β-Glucosidase cleaves pNPG → releases yellow para-nitrophenol
• Read absorbance at 405 nm over time (kinetic assay, 30 min)
• Compare: transconjugants vs. untransformed recipient vs. donor vs. blank

Technologies:

• Spark Plate Reader or PHERAstar FSX — kinetic absorbance at 405 nm in 96-round-axygen half-deep plates
• Reagent dispensing: Multiflo automated microplate dispenser for substrate addition
• Data: calculate specific activity in nmol pNP / min / mg total protein (protein concentration by Bradford assay at 595 nm on same reader)

5. Spatial Resolution of Bioprinting — Image Analysis

What is measured: The geometric precision of the Opentrons OT-2 deposition — do donor and recipient zones remain spatially segregated, and how sharp is the boundary?

• Scan plates before and after incubation
• In ImageJ: draw intensity line profiles across zone boundaries → measure zone width, boundary sharpness (µm), and bleed-through area
• Vary deposited volume (0.5 µL, 1 µL, 2 µL) and cell density (OD600 0.1–1.0) to optimize printing parameters

Technologies:

• Flatbed scanner (600–1200 dpi)
• ImageJ macro for automated line-profile extraction
• Python + matplotlib for plotting bleed-through as a function of print volume

Homework: Waters Part I — Molecular Weight

1.

By using the molecular weight calculator provided by the ExPASy portal, the molecular weight of the N-terminally tagged eGFP presented above was calculated to be MW_th = 28,006.60Da.

2.1.

From Figure 10.1 I chose two adjacent charge state peaks:

• m/zₙ = 903.7148
• m/zₙ₊₁ = 875.4421

Therefore:

• 903.7148 → z = 31
• 875.4421 → z = 32

2.2.

Based on the calculations of the previous segment the experimentally measured MW of eGFP is MW_exp approximately 27,984Da

2.3.

By applying the mathematical formula, the accuracy of the measurement is approximately 8.1x10^-4.

3.

The peaks in the zoomed-in region are not clearly separated, likely due to limited instrument resolution at this m/z range. Because the isotopic peaks are only partially resolved and somewhat noisy, it is difficult to directly determine the charge state from isotope spacing alone.

However, by counting the charge states from previously assigned peaks (moving toward higher m/z and decreasing the charge by one for each peak), the zoomed-in peak at m/z ≈ 1474 corresponds to approximately the 19 charge state.

Homework: Waters Part III — Peptide Mapping - primary structure

1.

Lysine (K): 20 residues. Arginine (R): 6 residues. Total K + R: 26.

2.

Using the ExPASy PeptideMass tool with the parameters in Figure 4: 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: 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, and 5.43 min. All exceed 10% relative abundance (threshold: ~1.2e6 counts, based on the tallest peak at 4.87 min with ~1.2e7 counts).

4.

No, the numbers do not exactly match. There are more peaks (21) than predicted peptides (19). This difference can be explained by incomplete ionization, co-eluting peptides, very small peptides not retained well on the column, missed detection due to low abundance, or peptides outside the 0.5–6 min window.

5.

The most abundant (base) peak in the spectrum is at m/z 525.76712. The charge state of the most abundant form of the peptide is 2+.

To calculate the singly charged mass, I use: [M+H]+ = (m/z × z) − (z − 1) × 1.0073

where:

• m/z = 525.76712
• z = 2
• 1.0073 Da = mass of a proton

525.76712 × 2 = 1051.53424
1051.53424 − 1.0073 = 1050.52694 Da

Final result:

[M+H]+ ≈ 1050.53 Da

6.

By comparing the experimentally determined singly charged mass
([M+H]+ ≈ 1050.53 Da) with the theoretical tryptic 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.

Mass error: 12.2 ppm. The error is slightly above 10 ppm, which is typically considered the upper confidence threshold for peptide identification.

7.

According to Figure 6, the sequence coverage for eGFP is 88%.

Homework: Waters Part IV — Oligomers

Homework: Waters Part V — Did I make GFP?

Yes, the data confirms the production of eGFP. The observed mass is in close agreement with the theoretical calculation (~28.0 kDa), with a relative mass error of 0.081% (807 ppm).