<aside> 🧬 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.

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Final Project Homework

<aside> <img src="/icons/exclamation-mark_orange.svg" alt="/icons/exclamation-mark_orange.svg" width="40px" /> Mandatory to MIT/Harvard Students, optional for Committed Listeners. Edited April 23 for clarity.

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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.
• 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.

Ans: Project Measurement Plan (help from chatgpt taken here to understand some topics)

In my final project, we will measure several critical aspects to verify successful DNA-based data encoding, storage, and retrieval:

1. Accuracy of DNA-Based Data Encoding and Decoding

• What We’ll Measure:

  The fidelity of digital data retrieval from synthetic DNA after encoding, synthesis, storage, and sequencing.

• How We’ll Measure It:

  The original binary data (e.g., a text file or image) will be compared with the output recovered through sequencing and decoding. We will quantify any discrepancies using error rates (e.g., bit error rate).

• Technology Used:

  • DNA Sequencing (ONT): The encoded DNA will be sequenced to retrieve the nucleotide string.
  • Bioinformatics Tools: Custom Python scripts will compare the sequenced output with the original data, detecting insertion/deletion/mismatch errors.

2. Structural Integrity and Quality of Synthesized DNA

• What We’ll Measure:

  The length and purity of the synthesized DNA sequences used for data storage.

• How We’ll Measure It:

  By analyzing the size and quality of the oligonucleotides before and after amplification.

• Technology Used:

  • Gel Electrophoresis: To confirm correct fragment length and detect any degradation or incorrect assembly.
  • Spectrophotometry (e.g., Nanodrop): To measure DNA concentration and assess purity (A260/A280 ratio).

3. Efficiency of DNA Amplification and Storage Stability

• What We’ll Measure:

  • PCR amplification yield.
  • DNA degradation over time or under various storage conditions (temperature, humidity).

• How We’ll Measure It:

  We will run time-based PCR amplifications and analyze the output intensity on agarose gels. Stability will be measured by comparing amplification success over time.

• Technology Used:

  • Quantitative PCR (qPCR): For precise amplification yield measurements.
  • Standard PCR and Gel Electrophoresis: For visual comparison of band intensity over time.

Optional Future Measurement: Mass Verification of Synthesized Constructs

• What We’ll Measure:

  If we create longer DNA constructs, we may validate the expected molecular weight.

• Technology Used:

  • Mass Spectrometry (e.g., MALDI-TOF): Can be used in a more advanced setting to confirm molecular weight of oligonucleotides.

Waters Homework

MIT_HTGAA_Homework.docx

<aside> <img src="/icons/exclamation-mark_orange.svg" alt="/icons/exclamation-mark_orange.svg" width="40px" /> Part 1 and 2 are mandatory for Committed Listeners and MIT/Harvard Students

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Experimental Investigations

1. Molecular weight – intact protein measurement
2. Primary amino acid sequence – peptide map

Optional Section

1. Protein structure and shape - native versus denatured protein measurement

Part 1: Molecular Weight

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).