Week 10 HW: Advanced Imaging & Measurement Technology

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.

  1. molecular validation (to confirm whether the Twist plasmids are correct and transformed properly) through the presence of the sender and receiver plasmid, correct insert size and sequence identity, then successful bacterial transformation
  2. functional signalling validation / AHL sensing ability through receiver activation threshold, GFP expression strength, dose-response curve, signal saturation
  3. spatial pattern formation to measure whether LuxI actually produces AHL through sender-generated AHL activity, effective AHL equivalent concentration, and signalling consistency between cultures
  4. ML/CAD dataset features through OT-2 plate maps, fluorescence reads, OD600 and metadata logs

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.

The project will measure genetic construct integrity, quorum-sensing response strength, sender-derived signalling output, and spatial cellular automata pattern formation.

  1. Plasmid integrity will be measured using colony PCR followed by agarose gel electrophoresis to confirm the expected insert sizes of the LuxI sender and LuxR-sfGFP receiver constructs. Sanger sequencing will then be used to verify promoter-RBS-CDS junctions, spacer regions, stop codons, and inducible promoter elements.

  2. Functional LuxR receiver performance will be quantified by exposing transformed receiver E. coli to a defined dilution series of 3-oxo-C6-HSL and measuring GFP fluorescence over time using a handmade fluorescence plate reader. OD600 measurements will also be collected for normalisation

  3. LuxI sender activity will be measured indirectly by collecting cell-free sender supernatant and applying it to the receiver strain, using the previously generated AHL-GFP calibration curve to infer effective signalling molecule concentration.

  4. Spatial pattern formation relevant to biological cellular automata will be measured using Opentrons OT-2 automated plate layouts combined with fluorescence time-lapse imaging. Metrics including activation radius, propagation velocity, neighbourhood response, and endpoint fluorescence distribution will be extracted and used as training data for ML-enhanced CAD prediction in Aim 2.

Waters Part I — Molecular Weight

  1. Calculated molecular weight

Compute pI/Mw - Results Theoretical pI/Mw (average)

Sequence: 10 20 30 40 50 60 MVSKGEELFT GVVPILVELD GDVNGHKFSV SGEGEGDATY GKLTLKFICT TGKLPVPWPT

    70         80         90        100        110        120

LVTTLTYGVQ CFSRYPDHMK QHDFFKSAMP EGYVQERTIF FKDDGNYKTR AEVKFEGDTL

   130        140        150        160        170        180

VNRIELKGID FKEDGNILGH KLEYNYNSHN VYIMADKQKN GIKVNFKIRH NIEDGSVQLA

   190        200        210        220        230        240

DHYQQNTPIG DGPVLLPDNH YLSTQSALSK DPNEKRDHMV LLEFVTAAGI TLGMDELYKL

EHHHHHH

Theoretical pI/Mw: 5.90 / 28006.60

2. Calculated molecular weight of the eGFP using the adjacent charge state approach described in the recitation

For my data I chose peaks at $m/z = 933.8044$ and $m/z = 966.0037$

$$966.0037 - 933.8044 = 32.1993$$

For $n$ and $n-1$:

$$\frac{MW}{n} - \frac{MW}{n-1} \approx \frac{MW}{n(n-1)}$$

$z = 30$ and $z = 29$:

$$\text{Expected spacing} \approx \frac{MW}{30 \times 29} \approx \frac{28000}{870} \approx 32.2$$

$m/z = 933.8044$ is $z = 30$

$m/z = 966.0037$ is $z = 29$

MW ($z = 30$):

$$MW = (30 \times 933.8044) - (30 \times 1.007825)$$ $$= 28,014.13 - 30.23$$ $$= 27,983.9\ \text{Da}$$

Super close to the theoretical MW of $28,006.60\ \text{Da}$ from the first step

3. accuracy of the measurement using the deconvoluted MW from 2.2 and the predicted weight of the protein from 2.1

$$\text{Accuracy} = \frac{|MW_{\text{experiment}} - MW_{\text{theory}}|}{MW_{\text{theory}}}$$ $$= \frac{|27,983.9 - 28,006.60|}{28,006.60}$$ $$= \frac{22.7}{28,006.60}$$ $$= 0.00081$$ $$= 0.081\%$$

Waters Part III — Peptide Mapping - primary structure

1. Lysines (K) and Arginines (R) are in eGFP

K R

MVSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYGKLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKQ HDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YIMADKQKNG IKVNFKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT LGMDELYKLE HHHHHH

K=20 R=6

2. Peptides generated from tryptic digestion of eGFP?

Theoretical pI: 5.90
Mw (average mass): 28006.60
Mw (monoisotopic mass): 27988.96

#MassPosition#MCModificationsPeptide Sequence
14472.1752170–2100HNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSK
22566.2931217–2390DHMVLLEFVTAAGITLGMDELYK
32437.26085–270GEELFTGVVPILVELDGDVNGHK
42378.257754–740LPVPWPTLVTTLTYGVQCFSR
51973.9062142–1570LEYNYNSHNVYIMADK
61503.659728–420FSVSGEGEGDATYGK
71266.578387–970SAMPEGYVQER
81083.4979240–2470LEHHHHHH
91050.5214115–1230FEGDTLVNR
10982.4952133–1410EDGNILGHK
11821.394081–860QHDFFK
12790.355275–800YPDHMK
13769.391347–530FICTTGK
14711.2944103–1080DDGNYK
15655.381398–1020TIFFK
16602.2780211–2150DPNEK
17579.3137128–1320GIDFK
18507.2925164–1670VNFK
19502.3235124–1270IELK

Predicted peptides = 19

  1. -5.

Observed LC peaks = ~19

Observed peaks are about the same as predicted

Main peptide peak = 525.76712

Spacing = 0.5 m/z, => most abundant charge state is 2+. The singly charged mass is 1050.52 Da

Charge = +2 [ M + H ] + [M+H] + = 1050.52145 Da [ M + H ] + [M+H] + = 1050.52438 Da

Identified peptide: FEGDTLVNR

the error is about 2.8 ppm

  1. Identify the peptide from PeptideMass

FEGDTLVNR has a theoretical monoisotopic mass of 1050.52143 Da

7. 88 is the percentage of the sequence that is confirmed by peptide mapping

Waters Part IV — Oligomers

7FU Decamer = 3.40 MDa

8FU Didecamer = 8 MDa

8FU 3-Decamer = 12 MDa

8FU 4-Decamer = 16 MDa

Waters Part V — Did I make GFP?

Yes!

ParameterTheoreticalObserved/Measured on Intact LC-MSPPM Mass ErrorMolecular Weight (kDa)
eGFP Monomer26,905 Da26,903 Da0.7 ppm26.90