<aside> <img src="/icons/exclamation-mark_brown.svg" alt="/icons/exclamation-mark_brown.svg" width="40px" /> This assignment focuses on designing, ordering, and assembling DNA constructs to support your final project goals. You will document your Benchling files, provide FASTA sequences for synthesis, and outline a precise experimental plan for cloning and validation. This is a suggested outline. You may do it in your own format.

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<aside> <img src="/icons/checkmark-line_green.svg" alt="/icons/checkmark-line_green.svg" width="40px" /> For MIT/Harvard students, if you would like additional feedback, please send a Word document to your TA mentor.

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1. DNA Design

<aside> <img src="/icons/dna_purple.svg" alt="/icons/dna_purple.svg" width="40px" /> For MIT/Harvard Students: Twist Ordering Form For Committed Listeners: Please speak with your node for DNA orders.

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1.1 Benchling Documentation

• Workspace Reference
  • Please include direct links or screenshots of your Benchling workspace.
  • Demonstrate plasmid maps and feature annotations (e.g., promoters, coding sequences, antibiotic markers, restriction sites).
  • Rationale: Explain why you chose specific elements (promoters, tags, reporters). As discussed in the Week 2 recitation notes, rational design involves ensuring promoter compatibility with your chosen host organism and matching reporter output to the downstream assay.
• Design Considerations
  • For each construct, note key details like GC content and predicted secondary structures that might affect assembly or expression efficiency.
  • Mention how these constructs align with your broader project aims (e.g., expression of a novel protein, sensor development, metabolic pathway engineering).

1.2 FASTA Files

• Submission-Ready Sequences
  • Provide the FASTA sequences for each designed construct (e.g., “construct_A_v1.fasta”, “construct_B_v2.fasta”).
  • Verify correctness by comparing Benchling’s in silico digest or alignment tool with your intended design.
• Multiple Variants
  • If creating multiple versions (e.g., different promoters or tags), label each FASTA file consistently so it’s clear which sequence belongs to which design variant.

1.3 Twist Order Requirements

• Order Summary
  • List the gene fragments or plasmid constructs you plan to have synthesized by Twist (or other providers).
  • Include final DNA length, GC content, and any relevant constraints (e.g., Twist’s max length for syntheses or minimal GC content restrictions).
  • Fill out the form, but please try making an account to see the process.
• Recitation Note Reminders
  • As mentioned in Week 2 and prior recitation sessions, ensure the design is free from restricted sites that might conflict with Twist’s synthesis pipeline. For example, some providers do not tolerate extreme GC stretches or strong hairpins.
  • Check for stop codons or frameshift issues if your insert is designed for protein expression.

2. Detailed Protocol

2.1 DNA Assembly and Cloning

1. Overview
   • This section outlines how you will assemble the constructs from the DNA designs above. Methods may include Gibson Assembly (Gibson et al., 2009), Golden Gate (Engler et al., 2008), or classical restriction-ligation approaches.
   • Reference relevant protocols from the recitation notes as well as standard molecular biology guides (e.g., Sambrook and Russell).
   • Also use the following resources: https://www.rcsb.org/, https://www.uniprot.org/
2. Step-by-Step Assembly
   1. Linearization or Fragment Preparation:
      • Enzymatically digest the backbone (if using restriction-ligation) or PCR-amplify your vector to create linear ends.
      • Purify fragments using a commercial kit (e.g., Qiagen).
   2. In-Fusion / Gibson / Golden Gate Reaction:
      • Prepare the reaction according to your chosen assembly protocol. For instance, for Gibson Assembly:
        • 50°C for 15–60 min (enzymatic assembly mix).
        • Reaction volumes typically range from 10–20 µL.
      • For Golden Gate:
        • Cyclic temperature program (e.g., 37°C for digestion, then 16°C for ligation) repeated 25–30 times.
   3. Transformation:
      • Transform chemically competent (e.g., DH5α) or electrocompetent cells with the assembled product.
      • Plate cells on the appropriate antibiotic selection medium.
   4. Colony Screening:
      • Pick individual colonies for colony PCR or direct plasmid miniprep.
3. Reagents and Materials
   • Assembly Mix: (e.g., NEB Gibson Master Mix or NEB Golden Gate Assembly Mix).
   • Competent Cells: E. coli DH5α, Top10, or a relevant strain recommended in Week 2 recitation notes.
   • Antibiotic Plates: LB-Agar supplemented with Amp, Kan, or your chosen selection marker.
   • PCR Primers: Designed with 20–30 bp overlap for Gibson or flanking type IIS sites for Golden Gate.