Week 2 HW: Read, Write & Edit

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HTGAA Homework 2

Part 1: Benchling & In-silico Gel Art See the Gel Art: Restriction Digests and Gel Electrophoresis protocol for details. Overview:

Make a free account at benchling.com Import the Lambda DNA. Simulate Restriction Enzyme Digestion with the following Enzymes:

  • EcoRI
  • HindIII
  • BamHI
  • KpnI
  • EcoRV
  • SacI
  • SalI

Create a pattern/image in the style of Paul Vanouse’s Latent Figure Protocol artworks.

Part 2: Gel Art - Restriction Digests and Gel Electrophoresis

Part 3: DNA Design Challenge

3.1. Choose your protein.

  • In recitation, we discussed that you will pick a protein for your homework that you find interesting. Which protein have you chosen and why? Using one of the tools described in recitation (NCBI, UniProt, google), obtain the protein sequence for the protein you chose.

Example from our group homework, you may notice the particular format — The example below came from UniProt

sp|P03609|LYS_BPMS2 Lysis protein OS=Escherichia phage MS2 OX=12022 PE=2 SV=1 METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLL EAVIRTVTTLQQLLT

3.2. Reverse Translate: Protein (amino acid) sequence to DNA (nucleotide) sequence.

  • The Central Dogma discussed in class and recitation describes the process in which DNA sequence becomes transcribed and translated into protein. The Central Dogma gives us the framework to work backwards from a given protein sequence and infer the DNA sequence that the protein is derived from. Using one of the tools discussed in class, NCBI or online tools (google “reverse translation tools”), determine the nucleotide sequence that corresponds to the protein sequence you chose above.

Example: Get to the original sequence of phage MS2 L-protein from its genome phage MS2 genome - Nucleotide - NCBI

  • Lysis protein DNA sequence
  • atggaaacccgattccctcagcaatcgcagcaaactccggcatctactaatagacgccggccattcaaacatgaggattacccatgtcgaagacaacaaagaagttcaactctttatgtattgatcttcctcgcgatctttctctcgaaatttaccaatcaattgcttctgtcgctactggaagcggtgatccgcacagtgacgactttacagcaattgcttacttaa

3.3. Codon optimization.

Once a nucleotide sequence of your protein is determined, you need to codon optimize your sequence. You may, once again, utilize google for a “codon optimization tool”. In your own words, describe why you need to optimize codon usage. Which organism have you chosen to optimize the codon sequence for and why?

Example from Codon Optimization Tool | Twist Bioscience while avoiding Type IIs enzyme recognition sites BsaI, BsmBI, and BbsI

  • Lysis protein DNA sequence with Codon-Optimization
  • ATGGAAACCCGCTTTCCGCAGCAGAGCCAGCAGACCCCGGCGAGCACCAACCGCCGCCGCCCGTTCAAACATGAAGATTATCCGTGCCGTCGTCAGCAGCGCAGCAGCACCCTGTATGTGCTGATTTTTCTGGCGATTTTTCTGAGCAAATTCACCAACCAGCTGCTGCTGAGCCTGCTGGAAGCGGTGATTCGCACAGTGACGACCCTGCAGCAGCTGCTGACCTAA

3.4. You have a sequence! Now what?

What technologies could be used to produce this protein from your DNA? Describe in your words the DNA sequence can be transcribed and translated into your protein. You may describe either cell-dependent or cell-free methods, or both.

3.5. [Optional] How does it work in nature/biological systems?

  • Describe how a single gene codes for multiple proteins at the transcriptional level.
  • Try aligning the DNA sequence, the transcribed RNA, and also the resulting translated Protein!!! See example below.
  • Example shows the biomolecular flow in central dogma from DNA to RNA to Protein] Special note that all “T” were transcribed into “U” and that the 3-nt codon represents 1-AA.

Part 4: Prepare a Twist DNA Synthesis Order

4.1. Create a Twist account, and Benchling account

4.2. Build Your DNA Insert Sequence

  • For example, let’s make a sequence that will make E. coli glow fluorescent green under UV light by constitutively (always) expressing sfGFP (a green fluorescent protein):

4.3. Select “Clonal Genes” option

  • For this demonstration, we’ll choose Clonal Genes. You’ll select clonal genes or gene fragments depending on your final project.

  • Historically, HTGAA projects using clonal genes (circular DNA) have reached experimental results 1-2 weeks quicker because they can be transformed directly into E. coli without additional assembly.

  • Gene fragments (linear DNA) offer greater design flexibility but typically require an assembly or cloning step prior to transformation. An advantage is If designed with the appropriate exonuclease protection, gene fragments can be used directly in cell-free expression.

4.4. Import your sequence

  • You just took an amino acid sequence of interest and converted it into DNA, codon optimized it, and built an expression cassette around it! Choose the Nucleotide Sequence option and Upload Sequence File to upload your FASTA file.

4.5. Choose Your Vector

  • Since we’re ordering a clonal gene, you will need to refer to Twist’s Vector Catalog to choose your circular backbone. You can think of this as taking your linear expression cassette for your protein of interest, and completing the rest of the circle!

  • The backbone confers many special properties like antibiotic resistance, an origin of replication, and more. Discuss with your node to decide on appropriate antibiotic options. At MIT/Harvard, you can use Ampicillin, Chloramphenicol, or Kanamycin resistance.

  • Twist vectors do not contain restriction sites near the insert fragment, so make sure to flank your design with cut sites if you are intending to extract this DNA insert fragment later.

  • For this demonstration, choose a Twist cloning vectors like pTwist Amp High Copy.

Part 5: DNA Read/Write/Edit

5.1 DNA Read

  1. What DNA would you want to sequence (e.g., read) and why? This could be DNA related to human health (e.g. genes related to disease research), environmental monitoring (e.g., sewage waste water, biodiversity analysis), and beyond (e.g. DNA data storage, biobank).

  2. In lecture, a variety of sequencing technologies were mentioned. What technology or technologies would you use to perform sequencing on your DNA and why? Also answer the following questions:

  3. Is your method first-, second- or third-generation or other? How so?

  4. What is your input? How do you prepare your input (e.g. fragmentation, adapter ligation, PCR)? List the essential steps.

  5. What are the essential steps of your chosen sequencing technology, how does it decode the bases of your DNA sample (base calling)?

  6. What is the output of your chosen sequencing technology?

5.2 DNA Write

  1. What DNA would you want to synthesize (e.g., write) and why? These could be individual genes, clusters of genes or genetic circuits, whole genomes, and beyond. As described in class thus far, applications could range from therapeutics and drug discovery (e.g., mRNA vaccines and therapies) to novel biomaterials (e.g. structural proteins), to sensors (e.g., genetic circuits for sensing and responding to inflammation, environmental stimuli, etc.), to art (DNA origamis). If possible, include the specific genetic sequence(s) of what you would like to synthesize! You will have the opportunity to actually have Twist synthesize these DNA constructs! :)

  2. What technology or technologies would you use to perform this DNA synthesis and why?

  • Also answer the following questions:

    1. What are the essential steps of your chosen sequencing methods?
    2. What are the limitations of your sequencing method (if any) in terms of speed, accuracy, scalability?

5.3 DNA Edit

  1. What DNA would you want to edit and why? In class, George shared a variety of ways to edit the genes and genomes of humans and other organisms. Such DNA editing technologies have profound implications for human health, development, and even human longevity and human augmentation. DNA editing is also already commonly leveraged for flora and fauna, for example in nature conservation efforts, (animal/plant restoration, de-extinction), or in agriculture (e.g. plant breeding, nitrogen fixation). What kinds of edits might you want to make to DNA (e.g., human genomes and beyond) and why?

  2. What technology or technologies would you use to perform these DNA edits and why?

  • Also answer the following questions:

    1. How does your technology of choice edit DNA? What are the essential steps?
    2. What preparation do you need to do (e.g. design steps) and what is the input (e.g. DNA template, enzymes, plasmids, primers, guides, cells) for the editing?
    3. What are the limitations of your editing methods (if any) in terms of efficiency or precision?