Week 2 HW: DNA Read, Write, and Edit

Part 0: Basics of Gel Electrophoresis

• Per instructions for this part, I attended the 02/10 lecture and 02/11. Additionally I attended all 3 Bootcamp sessions.

Part 1: Benchling & In-silico Gel Art

• Make a free account at benchling.com

Benchling Account Creation Confirmation

• Import the Lambda DNA

Benchling Phage Lambda DNA Import Confirmation_02.12.26

• Simulate Restriction Enzyme Digestion with the following Enzymes:

  • EcoRI

  Benchling EcoRI Enzyme Digest Confirmation

  • HindIII

  Benchling HindIII Enzyme Digest Confirmation

  • BamHI

  Benchling BamHI Enzyme Digest Confirmation

  • KpnI

  Benchling KpnI Enzyme Digest Confirmation

  • EcoRV

  Benchling EcoRV Enzyme Digest Confirmation

  • SacI

  Benchling SacI Enzyme Digest Confirmation

  • SalI

  Benchling SalI Enzyme Digest Confirmation

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

  

  Mind the Gap (Or a Most Wondrous Cave) ➕

Part 2: Gel Art - Restriction Digests and Gel Electrophoresis

Part 3: DNA Design Challenge

3.1 Choose your protein

• I chose the Mantis Fibroin 1 protein because for some reason when I received this assignment, my mind flipped to an insect protein, and then from there, a praying mantis. Upon further research, I was pleased with where my intuition lead me. The Mantis Fibroin 1 protein helps comprise the mantis’ ootheca, otherwise known as its egg casing. What’s fascinating about these proteins is that they create this coiled yet flexible foam-like structure around the mantis’ eggs. This protein piqued my interest, as it might have biomimetic potential. The Mantis Fibroin 1 protein is listed below ¹²:

tr|I3PM87|I3PM87_9NEOP Mantis fibroin 1 OS=Pseudomantis albofimbriata OX=627833 GN=MF1 PE=2 SV=1 MDSKMLCVSLLLAVFCLWYTEASPLEEKYGEKYGDMEEYQRGTEDSRAVINDHTAKVASQ SARGMVNKAKTTEAAARSNEQLSKDRQYYYREYLKKADYHKKKALEYEQLSAAENAKIAY HESKQKDWETKARESDVQCRDAEAKYEQSYTRSRELKRESIIAYVQAAMHHAEASGDHMK ADRAKDIARDMMRKAESLRGDASNHYQRSEEDKNKARSEKVKAHQNADNSQRHHTACRAY DQEGLKTRLSSKANMMRQIHSSLLAERSHSLAREDGLAADLSHKLAEELARMSEESGAIS KINSGEERGYSNKVRQDEVKAHELAVSKRMMGAEVADNSEMISLAQAKDGSLDEGENYKL STFYADDSTKNMLPDSRGQMSYGDE

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

• A translated Mantis Fibroin 1 protein nucleotide sequence of most likely codons is below, as well as evidence showing how I inserted the Mantis Fibroin 1 protein UniProt information into the reverse translation tool

atggatagcaaaatgctgtgcgtgagcctgctgctggcggtgttttgcctgtggtatacc gaagcgagcccgctggaagaaaaatatggcgaaaaatatggcgatatggaagaatatcag cgcggcaccgaagatagccgcgcggtgattaacgatcataccgcgaaagtggcgagccag agcgcgcgcggcatggtgaacaaagcgaaaaccaccgaagcggcggcgcgcagcaacgaa cagctgagcaaagatcgccagtattattatcgcgaatatctgaaaaaagcggattatcat aaaaaaaaagcgctggaatatgaacagctgagcgcggcggaaaacgcgaaaattgcgtat catgaaagcaaacagaaagattgggaaaccaaagcgcgcgaaagcgatgtgcagtgccgc gatgcggaagcgaaatatgaacagagctatacccgcagccgcgaactgaaacgcgaaagc attattgcgtatgtgcaggcggcgatgcatcatgcggaagcgagcggcgatcatatgaaa gcggatcgcgcgaaagatattgcgcgcgatatgatgcgcaaagcggaaagcctgcgcggc gatgcgagcaaccattatcagcgcagcgaagaagataaaaacaaagcgcgcagcgaaaaa gtgaaagcgcatcagaacgcggataacagccagcgccatcataccgcgtgccgcgcgtat gatcaggaaggcctgaaaacccgcctgagcagcaaagcgaacatgatgcgccagattcat agcagcctgctggcggaacgcagccatagcctggcgcgcgaagatggcctggcggcggat ctgagccataaactggcggaagaactggcgcgcatgagcgaagaaagcggcgcgattagc aaaattaacagcggcgaagaacgcggctatagcaacaaagtgcgccaggatgaagtgaaa gcgcatgaactggcggtgagcaaacgcatgatgggcgcggaagtggcggataacagcgaa atgattagcctggcgcaggcgaaagatggcagcctggatgaaggcgaaaactataaactg agcaccttttatgcggatgatagcaccaaaaacatgctgccggatagccgcggccagatg agctatggcgatgaa

Mantis Fibroin 1 UniProt Information Inserted into Reverse Translation Tool

3.3 Codon Optimization

• We need to optimize codon usage so a particular sequence can be expressed with greater fidelity, reliability, and efficiency in a host organism. I’ve chosen to optimze the codon sequence for Saccharomyces cerevisiae (baker’s yeast), because it:
• is commonly used as a host in biotechnology applications
• folds in a manner closer to insect protein folding
• is apparently easier to work with than mammalian cells

A codon optimized Mantis Fibroin 1 nucleotide sequence is shown below, as well as evidence showing how I showing how I inserted the Mantis Fibroin 1 protein nucleotide sequence information into the codon optimization tool

ATGGACAGTAAGATGTTATGTGTCTCCTTATTGTTGGCTGTTTTTTGTTTATGGTATACTGAAGCTTCCCCATTAGAAGAAAAGTATGGTGAAAAGTACGGTGACATGGAAGAGTACCAAAGAGGTACTGAAGATTCAAGAGCAGTTATTAACGATCATACTGCTAAAGTTGCTTCCCAATCCGCCAGAGGTATGGTTAATAAGGCTAAGACTACAGAAGCTGCTGCTAGAAGTAATGAACAATTATCTAAAGATAGACAATACTATTACAGAGAATATTTGAAAAAGGCTGATTATCATAAGAAGAAAGCTTTGGAATATGAACAGCTTTCAGCTGCTGAAAATGCAAAAATTGCTTATCATGAATCTAAACAAAAAGACTGGGAAACGAAAGCCAGAGAATCCGATGTTCAATGTCGTGATGCTGAAGCAAAATATGAACAATCTTACACAAGGTCCAGAGAACTGAAAAGGGAATCTATTATTGCTTATGTTCAAGCTGCTATGCATCATGCTGAAGCTAGCGGTGATCACATGAAAGCTGATAGAGCTAAAGATATCGCTAGAGATATGATGAGAAAGGCAGAATCCTTAAGGGGTGACGCTAGCAACCATTATCAGAGATCCGAAGAAGATAAGAATAAGGCCAGATCTGAAAAGGTTAAAGCTCATCAAAACGCTGATAATTCTCAAAGACATCATACTGCATGCAGAGCGTATGACCAAGAAGGTTTAAAGACGAGATTGAGCTCAAAAGCCAACATGATGAGACAAATTCACTCCTCACTACTGGCTGAAAGATCTCATTCATTAGCAAGAGAAGACGGTCTTGCGGCCGATTTATCACATAAGTTGGCTGAAGAATTAGCTAGAATGTCCGAAGAATCAGGTGCTATATCTAAAATAAACTCAGGTGAAGAAAGAGGCTATTCGAATAAAGTGAGACAAGATGAGGTTAAAGCACATGAATTGGCTGTTAGCAAAAGAATGATGGGTGCTGAAGTTGCTGATAATTCGGAGATGATTAGTTTGGCACAAGCTAAAGACGGTTCTTTAGATGAAGGTGAGAACTATAAATTATCCACTTTTTATGCAGACGATTCTACAAAAAATATGCTACCAGATTCTAGGGGTCAAATGTCTTACGGTGATGAA

Mantis Fibroin 1 nucleotide sequence codon optimized for Saccharomyces cerevisiae (baker’s yeast)

3.4: You have a sequence! Now what?

• Several cell-dependent technologies could be used for producing the codon-optimized Mantis Fibroin 1 protein. One such technology, a yeast system, has already been pursued in the previous steps of this section, as Saccharomyces cerevisiae (baker’s yeast) is a form of yeast. Bacterial systems, such as E. coli could also be used for producing the protein in host cell culture, although this would require different codon optimization. Other cell-dependent technologies could have included insect or mammalian-based systems, although I’m not sure of the value of expressing an insect-associated protein in another insect-based host (although this may be a failure of imagination on my part). As mentioned previously, mammalian systems could also be used, but apparently mammalian cells are more difficult to work with than bacterial or yeast-based hosts. Cell-free methods for producing the codon-optimized Mantis Fibroin 1 protein would involve breaking open a cell, extracting relevant ribosomes, enzymes, tRNAs, etc., and then taking these contents and combinining them with a DNA template (in this case our Mantis Fibroin 1 protein nucleotide sequence), energy sourcesm relevant amino acids, and a reaction buffer. There’s a time advantage of cell-free methods over cell-dependent methods of protein expression.

3.5 Optional: How does it work in nature/biological systems?

• From my research in answering this question, I think the answer is that post-transcriptional process called alternative splicing occurs, where non-coding mRNA (introns) are cut and removed, while coding regions (exons) remain ³. It’s pretty fascinating because this splicing can create several different types of mRNA molecules, and therefore different proteins. This increases the efficiency with which different proteins can be expressed within a particular organism.
• See below

Attempted Mantis Fibroin 1 Alignment

All Section 3 Prompts Listed Below

Part 4: Prepare a Twist DNA Synthesis Order

4.1: Create a Twist account, and Benchling account

Twist Account Creation

Benchling Account Creation Confirmation

4.2: Build Your DNA Insert Sequence

Original Sequence Insertion NOTE: Think I may’ve started off inserting the wrong sequence. This may have been potentially fixed when I inserted a sfGFP sequence from NCBI*

Codon Optimization NOTE: Think I may’ve started off inserting the wrong sequence. This may have been potentially fixed when I inserted a sfGFP sequence from NCBI*

Corrected NCBI Sequence Insertion

Corrected NCBI Sequence Codon Optimization

Start Codon Annotation

Stop Codon Annotation

Promoter BBa_J23106 Insertion

RBS Insertion

Coding Sequence Insertion

7x His Tag Insertion

Terminator BBa_B0015 Insertion

Sequence Linear View

• Downloaded Sequence FASTA file (via Mac OS TextEdit). See below:

  HQ873313 (codon optimized) TTTACGGCTAGCTCAGTCCTAGGTATAGTGCTAGCCATTAAAGAGGAGAAAGGTACCatgAGCAAAGGAGAAGAACTTT TCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGA AGGTGATGCTACAAACGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACACTT GTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAACGGCATGACTTTTTCAAGAGTG CCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGACCTACAAGACGCGTGCTGAAGTCAA GTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACAC AAACTCGAGTACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCA AAATTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCC TGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATG GTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAAcgtaaaggcgaggagctgt tcactggtgtcgtccctattctggtggaactggatggtgatgtcaacggtcataagttttccgtgcgtggcgagggtga aggtgacgcaactaatggtaaactgacgctgaagttcatctgtactactggtaaactgccggtaccttggccgactctg gtaacgacgctgacttatggtgttcagtgctttgctcgttatccggaccatatgaagcagcatgacttcttcaagtccg ccatgccggaaggctatgtgcaggaacgcacgatttcctttaaggatgacggcacgtacaaaacgcgtgcggaagtgaa atttgaaggcgataccctggtaaaccgcattgagctgaaaggcattgactttaaagaagacggcaatatcctgggccat aagctggaatacaattttaacagccacaatgtttacatcaccgccgataaacaaaaaaatggcattaaagcgaatttta aaattcgccacaacgtggaggatggcagcgtgcagctggctgatcactaccagcaaaacactccaatcggtgatggtcc tgttctgctgccagacaatcactatctgagcacgcaaagcgttctgtctaaagatccgaacgagaaacgcgatcatatg gttctgctggagttcgtaaccgcagcgggcatcacgcatggtatggatgaactgtacaaatgaCATCACCATCACCATC ATCACtaaCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTG AACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA

4.3: On Twist, Select the Genes Option

Selected Genes

4.4: Select the ‘Clonal Genes’ Option

Clonal Genes Selected see results in subsections 4.5 and 4.6 below

4.5: Import your sequence

Imported Sequence (Step 1)

Imported Sequence (Step 2)

4.6: Choose Your Vector

Chose Vector

Downloaded Construct

Imported sequence into Benchling and viewed resulting plasmid

All Section 4 Prompts Listed Below

Part 5: DNA Read/Write/Edit

5.1 DNA Read

• (i) What DNA would you want to sequence (e.g., read) and why?

• I want to sequence the DNA of the bdelloid rotifer Adineta genus, a microscopic-sized invertebrate that kind of looks like a worm. I’m fascinated by its ability to sustain cryptobiosis for thousands (in the case of a bdelloid rotifer thawed out in Russia in 2015, more than 24,000!!) of years. Transgenesis of the bdelloid rotifer’s cryptobiotic abilities in mammalian organisms could have profound impacts on the future of the species, specifically the ability for groups of homo sapiens or other future sapiens forms to engage in interstellar travel over large durations of time and space. More information on the bdelloid rotifer Adineta genus and its cryptobiotic abilities can be found in the footnote at the end of this sentence ⁴.

• (ii) In lecture, a variety of sequencing technologies were mentioned. What technology or technologies would you use to perform sequencing on your DNA and why?

  • I’d use Next-Generation Sequencing (NGS) on my DNA because it’s well-suited for transgenesis. It’s fast, high-resolution, and allows for massively parallel sequencing (if desired). More importantly, it’s highly precise, meaning it can pinpoint transgene locations within a host genome.

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

  • It’s a second-generation sequencing method, as it emerged in the 2000’s after the advent of Sanger sequencing in the 1970’s and before the advent of single-molecule sequencing in the 2010’s.

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

  • If the gene exists in an organism, the initial input is extracted genomic DNA from that particular organism. Otherwise, the initial input can be a plasmid (if the DNA’s already cloned) or complementary DNA (cDNA) if only mRNA is available. Essential preparation steps listed below (assuming gene exists in an organism):
    • Isolate or extract the gene; lyse cells or tissues from donor, remove proteins and contaminants
    • Fragment the isolated or extracted gene into fragments of apprxo. ~200-600bp
    • Convert ragged ends into blunt or sticky ends
    • Attach adapter sequences to ends of each fragment
    • Enrich fragments of the intended size (i.e., the size you want), removing any remaining small artifacts and excess adapters
    • Review fragement size distribution
    • Convert double-stranded library into single strands if necessary
    • Load into sequencing instrument

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

  • The essential NGS steps are listed below:
    • Preparation (see above)
    • Amplification: Many copies of each DNA fragment are created on flow cells. One end of each fragment sticks to a primer, gets copied to a complementary strand, and bends over to stick to a primer. This bridging repeats n^th times, forming amplified (hence the name) clusters of these identical grouped fragments
    • Sequencing: A polymerase enzymes adds 1 colored flourescent nucleotide to each strand in each cluster of grouped fragments. A camera takes a picture of recording the color for the given nucleotide (revealing its A, T, G, or C base) then chemicals are used to wash away any free-floating nucleotides that aren’t part of a given cluster.
    • Base Decoding/Base Calling: A computer analyzes these colored image clusters, assigning each cluster a sequence of bases based on its colors. This analysis then becomes a text string of DNA code, with confidence ratings per base sequence based on the resolution of the read ⁵

• What is the output of your chosen sequencing technology?

  • See above. NGS outputs a text string of DNA code, with confidence ratings per base sequence

5.2 DNA Write

• (i) What DNA would you want to synthesize (e.g., write) and why?)

  • See answer to 5.1 (i). I’m fascinated by the potential of transgenesis of the bdelloid rotifer Adineta’s cryptobiotic abilities for interstellar travel, and therefore, am very interested in reading and editing its sequence.

  NOTE: I found a bdelloid rotifer Adineta sequence in the link in the footnote. However, the raw FASTA information is so long that upon insertion into this webpage, it seemingly broke the webpage, or caused it to freeze up (pardon the unintentional cryptobiosis pun) ⁶

• (ii) What technology or technologies would you use to perform this DNA synthesis and why?

  • I’d use PCR amplification, chemical synthesis, and restriction enzymes and ligation to perform this DNA synthesis. I’d use PCR so I can make many copies of the original DNA, I’d use chemical synthesis so I can clone the DNA into a given plasmid, encapsulating the DNA for desired level expression in an appropiate vehicle, and I’d use restriction enzymes and ligation for precise synthesis.
  • What are the essential steps of your chosen sequencing methods?
    • PCR amplification:
      • Denaturation: Heat DNA so it breaks into stingle strands
      • Annealing: Cool the DNA, allowing primers to bind to sites in target gene
      • Extension: DNA polymerase adds nucleotides from the primer starting point, allowing each strand to fully copy
    • Chemical synthesis:
      • Deprotection: Remove protecting DMT group
      • Base Coupling: Add protected phosphoramidite nucleotide for phosphate linkage
      • Capping: Cap the chain to prevent errors
      • Oxidation: Stabilize phosphate triester bonds
    • Restriction enzymes and ligation:
      • Stitch oligos from prior chemical synthesis step into a complete gene for insertion into plasmid
      • Add flanking restriction enzymes at ends of oligos as needed
      • Clean the isolated DNA via gel extract
      • Use matching restriction enzymes to incubate the gene insert and its plasmid vector. This incubation process recognizes specific sequences in the gene insert and cut, creating blunt and sticky ends
      • Use DNA ligase enzyme to mix compatible ends
  • What are the limitations of your sequencing method (if any) in terms of speed, accuracy, scalability?
    • A scalability challenge is that it’s difficult to synthesize a sequence of more than 200nt via direct synthesis methods because at a certain point, too many errors accumulate in the synthesized sequence

5.2 DNA Edit

• (i) What DNA would you want to edit and why?
  • See answer to 5.1 (i). I’m fascinated by the potential of transgenesis of the bdelloid rotifer Adineta’s cryptobiotic abilities for interstellar travel, and therefore, am very interested in reading and editing its sequence.
• (ii) What technology or technologies would you use to perform these DNA edits and why?
  • I’d use CRISPR-Cas9 to perform these DNA edits because when performing transgenesis from an invertebrate to a mammalian vertebrate, it allows for non-random, precise insertion of large amounts of genetic data with reduced risk of unintended or off-target effects.
  • How does your technology of choice edit DNA? What are the essential steps?
    • CRISPR-Cas9 edits DNA through a multi-stage mechanism. This mechanism is broken down below:
      • Recognition: A single guide RNA (sgRNA) pairs with a Cas9 protein
      • This pair scans the genome for a 20bp DNA sequence if the sequence is next to a protospacer adjacent motif (PAM)
      • If there is a PAM next to the desired 20bp DNA sequence, the Cas9 makes a dobule-stranded break (DSB)
      • The DSB then triggers repair mechanisms (either non-homolgous end joining [NHEJ] or homology-directed repair [HDR]). These repair mechanisms allow the desired edited DNA to be incorporated into the sequence
  • 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?
    • Preparation:
      • Select target site(s)
      • Design and synthesize gRNA
      • Build DNA donor templates
      • Create mixture of recombinant Cas9 protein and purified gRNA
      • Preapre cells/embryos
    • Inputs to this process are gRNDA,Cas9 protein, donor DNA (usually linear templates), and a delivery vehicle (usually an injection buffer)
  • What are the limitations of your editing methods (if any) in terms of efficiency or precision?
    • HDR can have low efficiency in a transgenesis context
    • Off-target or unintended consequences can still occur
    • Need PAMs near target sequences for precise DSBs

All Section 5 Prompts Listed Below