Homework is based on data that will be generated in the Waters Immerse Lab in Cambridge, MA. Students will be characterizing green fluorescent protein (eGFP, a recombinant protein standard) structure (primary, secondary/tertiary) in the lab using liquid chromatography and mass spectrometry. Data generated in the lab will be available on-line for students working remotely.
Mandatory to MIT/Harvard Students, optional for Committed Listeners. Edited April 23 for clarity.
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.
I would love to attempt this question as my final project idea is heavily based on synthetic biology based detectables hence, yes I for sure will be detecting a biosensor , mostly a volatile organic compound like acetone from perspiration of a person, mainly for the better detection of metabolic disorders like diabetes , diabetic ketoacidosis I am thinking of creating a synthetic circuit that uses a logic gate like system (I am thinking something like conformational change- A riboswitch having a VOC specific aptamer) that detects the presence of the biomarker i.e. Acetone As the amount of volatile compounds present in sweat are relatively less, I will be aiming for high sensitivity mostly via aptamer optimization.
Please describe all of the elements you would like to measure, and furthermore describe how you will perform these measurements.
The chemical nature of Acetone is of small molecule. I aim to measure is the presence and concentration of acetone—a small molecule VOC—found in human sweat. The goal is to detect it using a synthetic riboswitch-based genetic circuit that is highly sensitive and specific to acetone. This riboswitch would include a custom-designed acetone-binding aptamer, which upon binding acetone, undergoes a conformational change to regulate gene expression downstream. This will mainly include measuring concentration of acetone using a quantitative reporting output like GFP, also a threshold which will be minimum amount of acetone detected also
What are the technologies you will use (e.g., gel electrophoresis, DNA sequencing, mass spectrometry, etc.)? Describe in detail.
I will definitely require cloning techniques and a lot of in-silico tools, also I think I will be employing PAGE and some spectroscopic techniques to validate the binding of biomarker to aptamer and analyze the conformational change.
Cloning and validation : The synthetic riboswitch circuit will be designed in benchling and synthesized via commercial services.Plasmid cloning mostly using Golden Gate Assembly will be used to construct the full genetic circuit. Validation of the construct will be performed using a sequencing technique, preferably first gen
For Reporter : A fluorescent reporter gene (e.g., GFP) or luminescent reporter (e.g., luciferase) will be incorporated downstream of the riboswitch.Fluorescence plate reader can be used to quantify the output response as a proxy for acetone concentration.
TX-TL and VOC exposure system: To test the circuit without the variability of live cells, a cell-free system can be used .These systems enable right environment and can quickly show whether the sensor . A sealed microfluidic can be used to expose the sensor system to known amounts of acetone vapor, mimicking physiological conditions (like sweat VOC release). Gas chromatography can be used to validate this concentration too
Data analysis tools: Tools like benchling, PyMol, RNAFold , Autodock vina to simulate and visualized the circuit, binding and conformation and Software like GraphPad Prism or Python (e.g., matplotlib, pandas) will be used for quantitative analysis of the signal vs. acetone concentration.
Part 1 and 2 are mandatory for Committed Listeners and MIT/Harvard Students
Experimental Investigations
Molecular weight – intact protein measurement
Primary amino acid sequence – peptide map
Optional Section
Protein structure and shape - native versus denatured protein measurement
We will be analyzing an eGFP standard onto a BioAccord LC-MS system to determine the molecular weight of intact eGFP and observe its charge state distribution in the denatured (unfolded) state. The conditions for LC-MS analysis of intact protein cause it to unfold and be detected in its denatured form (due to the solvents and pH used for analysis).
Questions
1.Based only on the predicted amino acid sequence of eGFP (see below), what is the calculated molecular weight? You can use an online calculator like the one here: https://web.expasy.org/compute_pi/