Week 2 HW: DNA Read, Write, & Edit

HOMEWORK 2

Part 1: Benchling & In-silico Gel Art

See this week’s lab protocol “Gel Art: Restriction Digests and Gel Electrophoresis” for details. Overview:

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

1. EcoRI
2. HindIII
3. BamHI
4. KpnI
5. EcoRV
6. SacI
7. SalI

• Create a pattern/image in the style of Paul Vanouse’s Latent Figure Protocol artworks.
• You might find Ronan’s website a helpful tool for quickly iterating on designs!

HOMEWORK RESULTS :)

1ST ATTEMPT

For my first attempt, I tried to form the phrase “Hi!”. It didn’t turn out as perfect as I imagined, but with practice, I hope to create more creative drawings.

2ND ATTEMPT

For my second attempt I tried to draw my own name in capital letters, “IAN”.

3RD ATTEMPT

For my third attempt I tried to draw the silhouette of an animal’s head.

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.

The protein I chose is …

Q68KI4 · NHX1_ARATH

Function: Acts in low affinity electroneutral exchange of protons for cations such as Na+ or K+ across membranes. Can also exchange Li+ and Cs+ with a lower affinity. Involved in vacuolar ion compartmentalization necessary for cell volume regulation and cytoplasmic Na+ detoxification. Required during leaves expansion, probably to stimulate epidermal cell expansion. Confers competence to grow in high salinity conditions.

FASTA sequence

sp|Q68KI4|NHX1_ARATH Sodium/hydrogen exchanger 1 OS=Arabidopsis thaliana OX=3702 GN=NHX1 PE=1 SV=2 MLDSLVSKLPSLSTSDHASVVALNLFVALLCACIVLGHLLEENRWMNESITALLIGLGTG VTILLISKGKSSHLLVFSEDLFFIYLLPPIIFNAGFQVKKKQFFRNFVTIMLFGAVGTII SCTIISLGVTQFFKKLDIGTFDLGDYLAIGAIFAATDSVCTLQVLNQDETPLLYSLVFGE GVVNDATSVVVFNAIQSFDLTHLNHEAAFHLLGNFLYLFLLSTLLGAATGLISAYVIKKL YFGRHSTDREVALMMLMAYLSYMLAELFDLSGILTVFFCGIVMSHYTWHNVTESSRITTK HTFATLSFLAETFIFLYVGMDALDIDKWRSVSDTPGTSIAVSSILMGLVMVGRAAFVFPL SFLSNLAKKNQSEKINFNMQVVIWWSGLMRGAVSMALAYNKFTRAGHTDVRGNAIMITST ITVCLFSTVVFGMLTKPLISYLLPHQNATTSMLSDDNTPKSIHIPLLDQDSFIEPSGNHN VPRPDSIRGFLTRPTRTVHYYWRQFDDSFMRPVFGGRGFVPFVPGSPTERNPPDLSKA

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]

Reverse Translation:

reverse translation of sp|Q68KI4|NHX1_ARATH Sodium/hydrogen exchanger 1 OS=Arabidopsis thaliana OX=3702 GN=NHX1 PE=1 SV=2 to a 1614 base sequence of most likely codons. atgctggatagcctggtgagcaaactgccgagcctgagcaccagcgatcatgcgagcgtggtggcgctgaacctgtttgtggcgctgctgtgcgcgtgcattgtgctgggccatctgctggaagaaaaccgctggatgaacgaaagcattaccgcgctgctgattggcctgggcaccggcgtgaccattctgctgattagcaaaggcaaaagcagccatctgctggtgtttagcgaagatctgttttttatttatctgctgccgccgattatttttaacgcgggctttcaggtgaaaaaaaaaacagttttttcgcaactttgtgaccattatgctgtttggcgcggtgggcaccattattagctgcaccattattagcctgggcgtgacccagttttttaaaaaactggatattggcacctttgatctgggcgattatctggcgattggcgcgatttttgcggcgaccgatagcgtgtgcaccctgcaggtgctgaaccaggatgaaaccccgctgctgtatagcctggtgtttggcgaaggcgtggtgaacgatgcgaccagcgtggtggtgtttaacgcgattcagagctttgatctgacccatctgaaccatgaagcggcgtttcatctgctgggcaactttctgtatctgtttctgctgagcaccctgctgggcgcggcgaccggcctgattagcgcgtatgtgattaaaaaactgtattttggccgccatagcaccgatcgcgaagtggcgctgatgatgctgatggcgtatctgagctatatgctggcggaactgtttgatctgagcggcattctgaccgtgtttttttgcggcattgtgatgagccattatacctggcataacgtgaccgaaagcagccgcattaccaccaaacatacctttgcgaccctgagctttctggcggaaacctttatttttctgtatgtgggcatggatgcgctggatattgataaatggcgcagcgtgagcgataccccgggcaccagcattgcggtgagcagcattctgatgggcctggtgatggtgggccgcgcggcgtttgtgtttccgctgagctttctgagcaacctggcgaaaaaaaaccagagcgaaaaaattaactttaacatgcaggtggtgatttggtggagcggcctgatgcgcggcgcggtgagcatggcgctggcgtataacaaatttacccgcgcgggccataccgatgtgcgcggcaacgcgattatgattaccagcaccattaccgtgtgcctgtttagcaccgtggtgtttggcatgctgaccaaaccgctgattagctatctgctgccgcatcagaacgcgaccaccagcatgctgagcgatgataacaccccgaaaagcattcatattccgctgctggatcaggatagctttattgaaccgagcggcaaccataacgtgccgcgcccggatagcattcgcggctttctgacccgcccgacccgcaccgtgcattattattggcgccagtttgatgatagctttatgcgcccggtgtttggcggccgcggctttgtgccgtttgtgccgggcagcccgaccgaacgcaacccgccggatctgagcaaagcg

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]

Codon Optimized sequence

NHX1_optimized_IDT ATGTTAGATTCTTTAGTTAGCAAATTGCCCTCACTCTCAACCTCTGACCACGCCAGCGTGGTTGCGCTGAACCTGTTTGTGGCGCTGCTGTGTGCCTGTATTGTGCTGGGCCACCTGCTGGAAGAAAACCGCTGGATGAATGAATCCATCACTGCGCTGCTGATCGGCCTGGGTACTGGTGTCACCATCCTGCTGATCAGTAAAGGCAAAGCTCCACCTGCTGGTGTTCTCTGAAGATCTGTTCTTTATCTATCTGCTGCCGCCGATCATCTTCAACGCCGGTTTCCAGGTGAAAAAGAAACAGTTCTTCCGTAATTCGTCACCATCATGCTGTTTGGTGCGGTAGGTACCATTATCAGCTGTACCATTATCAGCCTGGGTGTGACTCAGTTCTTCAAAAAACTGGATATCGGTACCTTTGACCTGGGTGATTATCTTGCGATTGGTGCGATCTTTGCTGCAACCGACAGTGTGTGCACCCTGCAGGTGCTGAACCAGGATGAAACCCCGCTGCTGTACAGCCTGGTGTTCGGTGAAGGTGTGGTGAACGATGCGACCTCGGTGGTGGTTTTAACGCCATTCAGAGCTTTGACCTGACCCATCTGAACCATGAAGCGGCGTTCCACCTGCTCGGCAACTTCCTGTACCTGTTCCTGCTGTCCACCCTGCTGGGTGCGGCGACCGGTCTGATCTCTGCCTATGTGATCAAGAAGCTGTATTTGGTCGTCACAGCACCGACCGCGAAGTTGCACTGATGATGCTGATGGCGTACCTGAGCTACATGCTGGCAGAGCTGTTTGACCTCAGTGGTATCCTGACCGTGTTCTTCTGCGGTATTGTCATGAGCCACTACACCTGGCATAACGTGACTGAAGCAGCCGTATCACCACCAAACACACCTTTGCCACCCTGTCGTTCTTGGCTGAAACCTTTATCTTCCTGTATGTCGGTATGGATGCGCTGGACATCGATAAGTGGCGCTCGGTAAGCGACACACCGGGTACCTCTATTGCGGTTAGCTCGATTCTGATGGGCCTGGTGATGGTAGGTCGTGCGGCGTTCGTGTTCCCGCTGTCGTTCTTGAGCAACCTGGCGGAGAAGAACCAGTCTGAGAAAATCAACTTCAACATGCAGGTGGTGATCTGGTGGTCTGGGCTGATGCGTGGTGCAGTCTCTATGGCCCTGGCCTACAACAAGTTTACCCGTGCAGGTCACACTGATGTACGTGGTAATGCGATTATGATCACCTCCACCATCACCGTGTGCCTGTTCAGCACCGTGGTGTTTGGCATGCTGACCAAACCGCTGATCAGCTACCTGCTGCCGCATCAGAATGCCACCACCAGCATGCTGTCTGATGACAACACGCCGAAATCTATTCACATTCCGCTGCTGGATCAGGACAGCTTTATTGAGCCGTCTGGTAACCACAATGTTCCACGTCCGGACAGCATTCGCGGTTTCCTGACCCGCCCGACCCGCACCGTGCACTACTATTGGCGTCAGTTTGATGACTCCTTCATGCGCCCGGTGTTTGGTGGTCGCGGCTTTGTGCCGTTTGTTCCGGGCTCCCCAACTGAGCGTAACCCGCCGGATCTGAGCAAAGCA

My answer:

Codon optimization is necessary because, although multiple codons can encode the same amino acid, each organism preferentially uses certain codons over others. For example, If a gene from one organism is expressed in a different host without optimization, rare codons may reduce translation efficiency, slow ribosome movement, decrease protein yield, or cause premature termination. The NHX1 coding sequence was optimized according to the codon usage preference of Escherichia coli, which was selected because it is one of the most widely used systems for recombinant protein expression due to its rapid growth, well - characterized genetics and availability of expression vectors and laboratory tools.

On the other hand, the codon optimization was performed using the IDT Codon Optimization Tool (Integrated DNA Technologies). During optimization, the tool adjusted synonymous codons to match E. coli codon bias while maintaining the original amino acid sequence. Additionally, to facilitate downstream cloning strategies, recognition sites for Type IIS restriction enzymes BsaI, BsmBI, and BbsI were avoided during the optimization process which ensures compatibility with Golden Gate assembly and prevents unwanted internal digestion of the gene sequence.

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.

My answer:

Cell-dependent protein expression

This option would clone the optimized NHX1 gene into an expression vector (plasmid) containing:

• A strong promoter.
• A ribosome binding site (RBS).
• A selectable marker (antibiotic resistance gene)
• A transcription terminator

The recombinant plasmid is then introduced into a host (Escherichia coli in this case), through transformation. Once inside the cell, the DNA sequence is transcribed, where RNA polymerase recognizes the promoter and synthesizes messenger RNA (mRNA) complementary to the coding strand of the DNA and translated where ribosomes bind to the mRNA and read the codons in triplets. Transfer RNAs (tRNAs) bring the corresponding amino acids, which are linked together through peptide bonds to form the NHX1 protein.

Part 4: Prepare a Twist DNA Synthesis Order

4.1. Create a Twist account and a 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): In Benchling, select New DNA/RNA sequence Give your insert sequence a name and select DNA with a Linear topology (this is a linear sequence that will be inserted into a circular backbone vector of our choosing).

The image above shows the Codon Optimized sequence of Q68KI4 · NHX1_ARATH.

Go through each piece of the given DNA sequences highlighted below (Promoter, RBS, Start Codon, Coding Sequence, His Tag, Stop Codon, Terminator) and paste the sequences into the Benchling file one after the other (replacing the coding sequence with your codon optimized DNA sequence of interest!). Each time you add a new piece of the sequence, make sure to annotate by right clicking over the sequence and creating an annotation that describes what each piece (e.g., Promoter, RBS, etc.) is.

Promoter (e.g. BBa_J23106): TTTACGGCTAGCTCAGTCCTAGGTATAGTGCTAGC

RBS (e.g. BBa_B0034 with spacers for optimal expression): CATTAAAGAGGAGAAAGGTACC

Start Codon: ATG

Coding Sequence (your codon optimized DNA for a protein of interest, sfGFP for example): AGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACACTTGTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGACCTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAACTCGAGTACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAA

7x His Tag (Let’s add a 7×His tag at the C-terminus of the protein to enable protein purification from E. coli): CATCACCATCACCATCATCAC

Stop Codon: TAA

Terminator (e.g. BBa_B0015): CCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA

Once you’ve completed this, click on Linear Map to preview the entire sequence. If you intend to have a TA review a sequence in the future, this is a good way to verify that all sections are annotated!

https://benchling.com/ian-teran-35/f_/91Ap236lfD-htgaa-2026/

Downloaded FASTA sequence of the construct:

AtNHX1_Ecoli_expression_construct TTTACGGCTAGCTCAGTCCTAGGTATAGTGCTAGCCATTAAAGAGGAGAAAGGTACCATGTTAGATTCTTTAGTTAGCAAATTGCCCTCACTCTCAACCTCTGACCACGCCAGCGTGGTTGCGCTGAACCTGTTTGTGGCGCTGCTGTGTGCCTGTATTGTGCTGGGCCACCTGCTGGAAGAAAACCGCTGGATGAATGAATCCATCACTGCGCTGCTGATCGGCCTGGGTACTGGTGTCACCATCCTGCTGATCAGTAAAGGCAAAGCTCCACCTGCTGGTGTTCTCTGAAGATCTGTTCTTTATCTATCTGCTGCCGCCGATCATCTTCAACGCCGGTTTCCAGGTGAAAAAGAAACAGTTCTTCCGTAATTCGTCACCATCATGCTGTTTGGTGCGGTAGGTACCATTATCAGCTGTACCATTATCAGCCTGGGTGTGACTCAGTTCTTCAAAAAACTGGATATCGGTACCTTTGACCTGGGTGATTATCTTGCGATTGGTGCGATCTTTGCTGCAACCGACAGTGTGTGCACCCTGCAGGTGCTGAACCAGGATGAAACCCCGCTGCTGTACAGCCTGGTGTTCGGTGAAGGTGTGGTGAACGATGCGACCTCGGTGGTGGTTTTAACGCCATTCAGAGCTTTGACCTGACCCATCTGAACCATGAAGCGGCGTTCCACCTGCTCGGCAACTTCCTGTACCTGTTCCTGCTGTCCACCCTGCTGGGTGCGGCGACCGGTCTGATCTCTGCCTATGTGATCAAGAAGCTGTATTTGGTCGTCACAGCACCGACCGCGAAGTTGCACTGATGATGCTGATGGCGTACCTGAGCTACATGCTGGCAGAGCTGTTTGACCTCAGTGGTATCCTGACCGTGTTCTTCTGCGGTATTGTCATGAGCCACTACACCTGGCATAACGTGACTGAAGCAGCCGTATCACCACCAAACACACCTTTGCCACCCTGTCGTTCTTGGCTGAAACCTTTATCTTCCTGTATGTCGGTATGGATGCGCTGGACATCGATAAGTGGCGCTCGGTAAGCGACACACCGGGTACCTCTATTGCGGTTAGCTCGATTCTGATGGGCCTGGTGATGGTAGGTCGTGCGGCGTTCGTGTTCCCGCTGTCGTTCTTGAGCAACCTGGCGGAGAAGAACCAGTCTGAGAAAATCAACTTCAACATGCAGGTGGTGATCTGGTGGTCTGGGCTGATGCGTGGTGCAGTCTCTATGGCCCTGGCCTACAACAAGTTTACCCGTGCAGGTCACACTGATGTACGTGGTAATGCGATTATGATCACCTCCACCATCACCGTGTGCCTGTTCAGCACCGTGGTGTTTGGCATGCTGACCAAACCGCTGATCAGCTACCTGCTGCCGCATCAGAATGCCACCACCAGCATGCTGTCTGATGACAACACGCCGAAATCTATTCACATTCCGCTGCTGGATCAGGACAGCTTTATTGAGCCGTCTGGTAACCACAATGTTCCACGTCCGGACAGCATTCGCGGTTTCCTGACCCGCCCGACCCGCACCGTGCACTACTATTGGCGTCAGTTTGATGACTCCTTCATGCGCCCGGTGTTTGGTGGTCGCGGCTTTGTGCCGTTTGTTCCGGGCTCCCCAACTGAGCGTAACCCGCCGGATCTGAGCAAAGCACATCACCATCACCATCATCACTAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA

4.3. On Twist, Select The “Genes” Option

4.4. 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.

4.5. 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.

4.6. 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. Click into your sequence and select download construct (GenBank) to get the full plasmid sequence:

Go back to your Benchling account. Inside of a folder, click the import DNA/RNA sequence button and upload the GenBank file you just downloaded.

WOW! :)

Part 5: DNA Read/Write/Edit

5.1 DNA Read (i) 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).

I would sequence the cry4Ba gene from Bacillus thuringiensis isolates in the field, the promoter and regulatory regions controlling cry4Ba expression and comparable cry4 family homologs from different strains because I would like to understand the genetic diversity of cry4Ba which would be useful to explore new methods to improve efficacy against mosquito larvae, reveal natural sequence variation influencing toxicity and assist in environmental monitoring of Bt toxin dissemination.

(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? Also answer the following questions:

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

The technology I would use is Illumina short-read sequencing which is a second generation sequencing method. It provides high accuracy, cost effectiveness and is well suited to bacterial genes.

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

• DNA extraction
• Fragmentation (to ~300 bp)
• Adapter ligation
• PCR enrichment
• Library quantification & pooling

The input is Genomic DNA from Bacillus thuringiensis cultures.

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

• Sequencing-by-synthesis.
• Each base is read by fluorescently labeled nucleotides incorporated one at a time.
• Signals are captured and used for base calling.

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

FASTQ files of read sequences and paired reads that can be aligned to reference genomes.

5.2 DNA Write (i) 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! :)

I would design and synthesize a codon-optimized cry4Ba gene for high toxin expression in a chosen bacterial host, variant versions with enhanced insecticidal activity and chimeric constructs combining parts of different Cry proteins to improve biological control of mosquitoes, increase production yield in recombinant strains and make toxin variants tailored to resistant insect populations.

cry4Ba gene DNA sequence:

ATGATGAATTCTGGTTATCCTTTAGCTAATGATTTACAAGGTTCTATGAAAAATACTAATTATAAAGATTGGTTAGCTATGTGTGAAAATAATCAACAATATGGTGTTAATCCTGCTGCTATTAATTCTTCTTCTGTTTCTACTGCTTTAAAAGTTGCTGGTGCTATTTTAAAATTTGTTAATCCTCCTGCTGGTACTGTTTTAACTGTTTTATCTGCTGTTTTACCTATTTTATGGCCTACTAATACTCCTACTCCTGAACGTGTTTGGAATGATTTTATGACTAATACTGGTAATTTAATTGATCAAACTGTTACTGCTTATGTTCGTACTGATGCTAATGCTAAAATGACTGTTGTTAAAGATTATTTAGATCAATATACTACTAAATTTAATACTTGGAAACGTGAACCTAATAATCAATCTTATCGTACTGCTGTTATTACTCAATTTAATTTAACTTCTGCTAAATTACGTGAAACTGCTGTTTATTTTTCTAATTTAGTTGGTTATGAATTATTATTATTACCTATTTATGCTCAAGTTGCTAATTTTAATTTATTATTAATTCGTGATGGTTTAATTAATGCTCAAGAATGGTCTTTAGCTCGTTCTGCTGGTGATCAATTATATAATACTATGGTTCAATATACTAAAGAATATATTGCTCATTCTATTACTTGGTATAATAAAGGTTTAGATGTTTTACGTAATAAATCTAATGGTCAATGGATTACTTTTAATGATTATAAACGTGAAATGACTATTCAAGTTTTAGATATTTTAGCTTTATTTGCTTCTTATGATCCTCGTCGTTATCCTGCTGATAAAATTGATAATACTAAATTATCTAAAACTGAATTTACTCGTGAAATTTATACTGCTTTAGTTGAATCTCCTTCTTCTAAATCTATTGCTGCTTTAGAAGCTGCTTTAACTCGTGATGTTCATTTATTTACTTGGTTAAAACGTGTTGATTTTTGGACTAATACTATTTATCAAGATTTACGTTTTTTATCTGCTAATAAAATTGGTTTTTCTTATACTAATTCTTCTGCTATGCAAGAATCTGGTATTTATGGTTCTTCTGGTTTTGGTTCTAATTTAACTCATCAAATTCAATTAAATTCTAATGTTTATAAAACTTCTATTACTGATACTTCTTCTCCTTCTAATCGTGTTACTAAAATGGATTTTTATAAAATTGATGGTACTTTAGCTTCTTATAATTCTAATATTACTCCTACTCCTGAAGGTTTACGTACTACTTTTTTTGGTTTTTCTACTAATGAAAATACTCCTAATCAACCTACTGTTAATGATTATACTCATATTTTATCTTATATTAAAACTGATGTTATTGATTATAATTCTAATCGTGTTTCTTTTGCTTGGACTCATAAAATTGTTGATCCTAATAATCAAATTTATACTGATGCTATTACTCAAGTTCCTGCTGTTAAATCTAATTTTTTAAATGCTACTGCTAAAGTTATTAAAGGTCCTGGTCATACTGGTGGTGATTTAGTTGCTTTAACTTCTAATGGTACTTTATCTGGTCGTATGGAAATTCAATGTAAAACTTCTATTTTTAATGATCCTACTCGTTCTTATGGTTTACGTATTCGTTATGCTGCTAATTCTCCTATTGTTTTAAATGTTTCTTATGTTTTACAAGGTGTTTCTCGTGGTACTACTATTTCTACTGAATCTACTTTTTCTCGTCCTAATAATATTATTCCTACTGATTTAAAATATGAAGAATTTCGTTATAAAGATCCTTTTGATGCTATTGTTCCTATGCGTTTATCTTCTAATCAATTAATTACTATTGCTATTCAACCTTTAAATATGACTTCTAATAATCAAGTTATTATTGATCGTATTGAAATTATTCCTATTACTCAATCTGTTTTAGATGAAACTGAAAATCAAAATTTAGAATCTGAACGTGAAGTTGTTAATGCTTTATTTACTAATGATGCTAAAGATGCTTTAAATATTGGTACTACTGATTATGATATTGATCAAGCTGCTAATTTAGTTGAATGTATTTCTGAAGAATTATATCCTAAAGAAAAAATGTTATTATTAGATGAAGTTAAAAATGCTAAACAATTATCTCAATCTCGTAATGTTTTACAAAATGGTGATTTTGAATCTGCTACTTTAGGTTGGACTACTTCTGATAATATTACTATTCAAGAAGATGATCCTATTTTTAAAGGTCATTATTTACATATGTCTGGTGCTCGTGATATTGATGGTACTATTTTTCCTACTTATATTTTTCAAAAAATTGATGAATCTAAATTAAAACCTTATACTCGTTATTTAGTTCGTGGTTTTGTTGGTTCTTCTAAAGATGTTGAATTAGTTGTTTCTCGTTATGGTGAAGAAATTGATGCTATTATGAATGTTCCTGCTGATTTAAATTATTTATATCCTTCTACTTTTGATTGTGAAGGTTCTAATCGTTGTGAAACTTCTGCTGTTCCTGCTAATATTGGTAATACTTCTGATATGTTATATTCTTGTCAATATGATACTGGTAAAAAACATGTTGTTTGTCAAGATTCTCATCAATTTTCTTTTACTATTGATACTGGTGCTTTAGATACTAATGAAAATATTGGTGTTTGGGTTATGTTTAAAATTTCTTCTCCTGATGGTTATGCTTCTTTAGATAATTTAGAAGTTATTGAAGAAGGTCCTATTGATGGTGAAGCTTTATCTCGTGTTAAACATATGGAAAAAAAATGGAATGATCAAATGGAAGCTAAACGTTCTGAAACTCAACAAGCTTATGATGTTGCTAAACAAGCTATTGATGCTTTATTTACTAATGTTCAAGATGAAGCTTTACAATTTGATACTACTTTAGCTCAAATTCAATATGCTGAATATTTAGTTCAATCTATTCCTTATGTTTATAATGATTGGTTATCTGATGTTCCTGGTATGAATTATGATATTTATGTTGAATTAGATGCTCGTGTTGCTCAAGCTCGTTATTTATATGATACTCGTAATATTATTAAAAATGGTGATTTTACTCAAGGTGTTATGGGTTGGCATGTTACTGGTAATGCTGATGTTCAACAAATTGATGGTGTTTCTGTTTTAGTTTTATCTAATTGGTCTGCTGGTGTTTCTCAAAATGTTCATTTACAACATAATCATGGTTATGTTTTACGTGTTATTGCTAAAAAAGAAGGTCCTGGTAATGGTTATGTTACTTTAATGGATTGTGAAGAAAATCAAGAAAAATTAACTTTTACTTCTTGTGAAGAAGGTTATATTACTAAAACTGTTGATGTTTTTCCTGATACTGATCGTGTTCGTATTGAAATTGGTGAAACTGAAGGTTCTTTTTATATTGAATCTATTGAATTAATTTGTATGAATGAATAA

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

I would use high throughput chemical DNA synthesis combined with assembly methods such as Gibson Assembly because chemical oligonucleotide synthesis (phosphoramidite chemistry) is the standard technology to produce short DNA fragments with controlled sequence and high purity and for this reason, they can be assembled into the full length cry4Ba gene using Gibson Assembly which joins overlapping oligonucleotides in a single reaction. I would choose this combination because it enables accurate synthesis of long genes, allows codon optimization for different expression hosts and supports easy modular design.

Also answer the following questions:

1. What are the essential steps of your chosen sequencing methods?

• Oligo synthesis
• Purification
• Gene assembly
• Cloning into an expression vector

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

• Cost increases with length.
• Errors may occur during oligo synthesis.
• Requires verification.

5.3 DNA Edit (i) 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?

I would edit the cry4Ba coding sequence to enhance toxicity or stability, regulatory elements to improve expression in non-Bt hosts and domains to broaden target specificity. The goal of this DNA Edit would be to develop improved insecticides or novel delivery systems.

(ii) What technology or technologies would you use to perform these DNA edits and why?

I would use CRISPR/Cas9 because it allows precise modification of DNA by using a guide RNA (gRNA) that directs the Cas9 nuclease to a specific sequence within the cry4Ba gene and it is highly specific, relatively easy to design, efficient in bacteria and also scalable for generating multiple toxin variants. On the other hand, If I wanted to introduce small point mutations to improve toxin activity or stability without creating double-strand breaks I would use CRISPR base editors enabling single nucleotide changes with greater precision and lower risk of unwanted insertions or deletions.

Also answer the following questions:

1. How does your technology of choice edit DNA? What are the essential steps?

CRISPR/Cas9 edits DNA by creating a targeted double strand break at a specific sequence defined by a designed guide RNA (gRNA) that would be complementary to the cry4Ba locus as it is computationally designed to match the desired target site and cloned or synthesized. Then, the Cas9 nuclease and gRNA are delivered into Bacillus cells via plasmid transformation. Once inside the cell, the gRNA directs Cas9 to the target sequence, where Cas9 introduces a precise cut in the DNA and the cell’s natural DNA repair mechanisms then repair the break either through non-homologous end joining or homologous recombination if a donor DNA template containing desired modifications is provided. Finally, edited colonies are screened and verified by PCR and sequencing to confirm the intended modification.

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?

• Design guide RNA targeting cry4Ba.
• Cas9 delivery vector or ribonucleoprotein.
• Editing template with desired mutations.
• Bacterial host cells.

3. What are the limitations of your editing methods (if any) in terms of efficiency or precision?

• Off - target activity (needs careful design).
• Editing efficiency depends on repair pathways.
• Delivery methods vary in success.