Advanced Imaging & Measurement

• Simulated GFP production (Aim 1). Activation of the AND-gate circuit's output layer is tracked in silico by modeling GFP reporter fluorescence across a 0–100 nM range of the Z₂ AHL inducer, generating a sigmoidal dose-response curve in Python/Matplotlib. The target: output below 5% of maximum in OFF states and above 90% in the (AHL HIGH, SRP HIGH) ON state.
• Population dynamics and auxotrophic interdependence (Aim 1). Growth curves tracking monoculture extinction versus stable co-culture steady states, computed from the six-equation Monod ODE system. The interior fixed point (n* ≈ 0.629, Re(λ_max) ≈ −0.215) and cascade extinction time under single-strain removal are the key quantitative outputs.
• Kill switch activation and off-target effects (Aim 2). Specific triggering of the MazE/MazF biocontainment module under low-phosphorus conditions, screened for unintended metabolic byproducts by MALDI-TOF and HPLC.
• Plasmid stability and biomass (Aim 2). Consortium viability under high-competition solid-state fermentation conditions — target: 10⁸–10⁹ CFU/g without loss of engineered circuits.
• Fecal coliform reduction (Aims 2–3). Concentration of fecal coliforms in multi-stress microcosms — target: greater than 3-log reduction from baseline (from the SEREMI-documented 54,000 NMP/100 mL).
• Phosphorus and H₂S sequestration (Aims 2–3). Environmental concentrations of soluble reactive phosphorus and hydrogen sulfide — targets: greater than 60% phosphorus reduction and greater than 80% H₂S reduction relative to initial conditions.
• Ecological resilience (Aims 2–3). Shannon entropy of microbial community composition in microcosms under operational stress, from 16S rRNA amplicon sequencing — the consortium should not collapse native biodiversity below the baseline Shannon entropy of undisturbed Budi sediments.

• Python / SciPy / Matplotlib. ODE integration (scipy.integrate.solve_ivp, RK45), Jacobian eigenvalue analysis (SymPy + scipy.linalg.eigvals), AND-gate Hill function modeling (NumPy). All Aim 1 computational deliverables.
• Benchling. In silico genetic circuit design — pFP-A, pFP-B, pFP-C annotated plasmids, virtual restriction digest, R-M shielding verification.
• MALDI-TOF mass spectrometry (UFRO-BIOREN). Confirmation that the AND-gate PhoA circuit produces the correct protein under inducing conditions; screening for off-target proteins when the MazE/MazF kill switch activates. MALDI-TOF provides rapid intact protein identification by molecular weight without chromatographic separation — the same technology demonstrated this week with eGFP.
• HPLC and HPLC-MS/MS (UFRO-BIOREN). Quantification of PhoA enzymatic output (alkaline phosphatase activity assay by pNPP substrate), screening for unintended metabolic byproducts, and confirmation of Microcin J25 lasso peptide production by Strain A. HPLC-MS/MS provides the secondary stringent toxin screening required before any organism enters a non-sterile environment.
• MPN coliform kits. Standard NMP/100 mL quantification of fecal coliform reduction in microcosm experiments. The regulatory baseline is 1,000 NMP/100 mL (NCh 1333); the documented contamination level at Puerto Domínguez was 54,000 NMP/100 mL.
• H₂S and SRP sensors. Electrochemical H₂S sensors and colorimetric SRP kits (ascorbic acid-molybdate method) for real-time tracking of remediation efficiency in microcosms.
• 16S rRNA amplicon sequencing. V3-V4 hypervariable region sequencing on Illumina MiSeq platform; Shannon entropy calculated from OTU tables using QIIME2. Baseline community composition from published studies of comparable Budi sediment samples.
• CRISPR/Cas9. Gene knockout verification (ΔhisD, ΔtrpB, ΔleuB) by colony PCR and Sanger sequencing of the deletion loci in Aim 2.
• Gibson Assembly. Physical construction of pFP-A, pFP-B, pFP-C plasmids from PCR-amplified inserts; confirmed by restriction digest gel electrophoresis and Sanger sequencing.

The eGFP sequence (246 amino acids including the His-tag linker LEHHHHHH) was submitted to ExPASy ProtParam and cross-validated with the PeptideMass tool (confirmed in the PeptideMass output header: Theoretical pI: 5.90 / Mw average mass: 28006.60 / Mw monoisotopic mass: 27988.96).

The difference between average and monoisotopic mass (~17.6 Da) reflects the contribution of naturally occurring heavy isotopes (¹³C, ¹⁵N, ²H) averaged across all atoms in the protein. At 28 kDa, the instrument resolves the monoisotopic mass — the mass of the most abundant isotopologue — rather than the average mass.

Figure 1 · Mass spectrum of intact eGFP · Waters Xevo G3 LC-MS (30,000 resolution) · Charge state envelope spanning m/z 700–1100 · Inset: zoomed peak at ~1473 m/z

Two adjacent peaks were selected from the denatured eGFP charge state envelope: m/z = 933.7349 (charge state n) and m/z = 903.7148 (charge state n+1).

Step 1: Determine z for each adjacent pair

Step 2: Determine MW from m/z and z

Step 3: Accuracy of measurement

The charge state of the zoomed peak at m/z ≈ 1473 can be determined by calculation, though resolving individual isotope peaks by eye requires care. From the intact MW of 27,983 Da and the observed m/z:

The inset in Figure 1 shows isotope peaks separated by approximately 0.05–0.15 m/z. At the instrument's 30,000 resolution and m/z ≈ 1473, the resolving power is approximately 1473/30,000 ≈ 0.049 Da — at the edge of resolving individual isotopes spaced 0.053 apart. The charge state of z = 19 is consistent with the observed peak pattern and the calculated MW.

A native protein maintains its three-dimensional fold through non-covalent interactions — hydrogen bonds, hydrophobic packing, electrostatic contacts, and van der Waals forces. These interactions compact the structure and, critically for mass spectrometry, determine how many proton-accepting sites are accessible to solvent. In the native state, many basic residues (Lys, Arg, His) are buried in the hydrophobic core or engaged in salt bridges, reducing their ability to acquire protons during electrospray ionization.

When a protein unfolds — by denaturing solvent (acetonitrile, methanol, low pH) — the polypeptide chain extends fully, exposing all basic residues to solvent and dramatically increasing the number of protons the molecule can accept. This produces a higher average charge state and a broader charge state distribution shifted toward lower m/z values in the denatured spectrum.

In Figure 2, the denatured eGFP spectrum (top) shows a broad envelope of high-charge states centered around m/z 800–1000 — consistent with 30+ protons on a fully extended 246-residue chain. The native eGFP spectrum (bottom) shows a narrow distribution at higher m/z (lower charge states), consistent with a compact folded structure where fewer basic residues are accessible. The shift to higher m/z and narrower distribution is the direct spectroscopic signature of tertiary structure.

From the native eGFP spectrum (Figure 3), the peak at ~2800 m/z corresponds to a low charge state of the folded protein. Using the intact MW of 27,983 Da:

The charge state is z = 10. This low charge state is expected for native eGFP: the compact β-barrel fold buries most basic residues, limiting proton uptake during electrospray. The narrow, symmetrical peak shape in the inset of Figure 3 confirms a single well-defined conformational state — the native fold — rather than the broad, heterogeneous distribution seen in the denatured spectrum.

Trypsin cleaves after Lys (K) and Arg (R) residues. Counting from the eGFP sequence:

The exact count is confirmed by the PeptideMass output below (19 peptides >500 Da covering 90.7% of the sequence).

The chromatographic peak at 2.78 min shows a dominant charge state at m/z = 525.76. From the isotope peak spacing in the Figure 5b inset:

Matching [M+H]⁺ = 1050.51 Da against the PeptideMass table: the closest entry is FEGDTLVNR (residues 115–123) with theoretical [M+H]⁺ = 1050.52149 Da (monoisotopic).

5.3 ppm is well within the typical accuracy specification of the Waters BioAccord LC-MS system (<5–15 ppm), confirming correct peptide identification.

Fragment Ion Calculator · FEGDTLVNR · pI 4.37 · [M+H]⁺ = 1050.52149 Da · [M+2H]²⁺ = 525.76441 Da · b/y ion series confirmed

Amino acid coverage map · eGFP tryptic digest · BioAccord LC-MS · 90.7% sequence coverage · Uncovered regions: mvsk (N-term) and aevk (region 116–119)

Keyhole Limpet Hemocyanin (KLH) exists in multiple oligomeric assemblies in solution. Using the subunit masses from Table 1 (7FU = 340 kDa, 8FU = 400 kDa), the expected masses for each oligomeric species are: