Week 2 HW: DNA design

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Author: Dr. Michellie Hernandez, MD Assisted by Kernel AI and ChatGPT for tutorial use of Kernel and broaden understanding. Content ideation and creation was done without AI and solely done by Dr. Michellie Hernandez

Release date: September 27, 2026

Part 0: Set up in Kernel

Victoria Makerspace Node Username: htgaa2@makerspace.ca Name of Repository in All repositories folder: Tfh_Michellie_Hernandez_test

Construct: Tfh cell simulator (IL-4, IL-21 and CD40-ligand Combination plasmid)

Part 1: Define your expression target

What molecule are you expressing?

  • Cytokine Interleukin-4 (IL-4)
  • Interleukin-21 (IL-21)
  • CD40-Ligand

What is the sequence?

What host or chassis?

  • Mammalian Cell-free system

Where does the product need to go?

  • Combined plasma will go in a cytoplasm in a cell-free system that needs to be secreted from the cell-free system

Part 2: Design your genetic parts

Vector context- Backbone appropriate to host

  • Part name: Signal regulatory region
  • Why? This part is a Regulatory DNA element that will respond to an external signal thus is a Signal-responsive regulator

Transcription start - Promoter (+ enhancer / intron if used)

  • Part name: Short HsEef1a1 promoter
    • From Kernel part Repository: Asimov Public
  • Why? Promotes DNA transcription in homo sapiens; Host: Mammalian cell-free system

Translation start - 5’ UTR with Kozak

  • Part name: HBA1 5’UTR
    • From Kernel part Repository: Mariana Diaz Luna_William&Mary
  • Why? Provides correct initiation of the translation.

Targeting - Signal peptide (secretion), tag

  • Part name: Signal peptide (sig-peptide)
    • From Homosapiens IL-4 gene from GenBank
  • Why? Tags the proteins in the Golgi apparatus to signal that proteins will be placed in vesicles for secretion.

Coding sequence - CDS (+start +stop)

  • Part name: 13677 bp
    • From Homo Sapien Gene sequence of IL-4 (8693 bp) from GenBank
    • From Homo Sapien mRNA of IL-21 (3137 bp)from GenBank
    • From Homo Sapien Gene sequence of CD40-Ligand mRNA (1852 bp) from GenBank:
  • Why? Provides the mRNA sequences of IL-4, IL-21 and CD40-Ligand each with appropriate start and stop codon for translation.

Transcription stop Terminator + Poly(A) signal

  • Part name: HBB 3’UTR
    • From Kernel part Repository: Mariana Diaz Luna_William&Mary
  • Part name: Poly(A) tail
    • From Kernel part Repository: Mariana Diaz Luna_William&Mary
  • Why for both transcription stop parts? Assures the termination of the translation.

Part 3: Assemble your construct

Figure 1 above

This construct is a mammalian cell-free system plasmid providing the substrates for the LF-OOAC organ-on-a-chip maintenance such as cytokines (IL-4 and IL-21) and costimulating CD40-Ligand that are normally produced in Follicular T helper cells (Tfh cells) as part of the HOPE factory workflow.

Part 4: Optimize and quality-check

  • Optimization of Codons: portion of IL-4 without stop codons in sequence Michellie Hernandez

ATC,GTT,AGC,TTC,TCC,ACT,AAT,TGC,CTC,ACA,TTG,TCA,CTG,CAA,ATC,GAC,ACC,TAT,TGG,GTC,TCA,CCT,CCC,AAC,TGC,TTC,CCC,CTC,TGT,TCT,TCC,TGC,CAT,GTG,CCG,GCA,ACT,TTG,TCC,ACG,GAC,ACA,AGT,GCG,ATA,TCA,CCT,TAC,AGG,AGA,TCA,TCA,AAA,CTT,ACA,GCC,TCA,CAG,AGC,AGA,AGG,GTA,CCT,ATC,TGG,CAC,CAT,CTC,TCC,AGA,TGT,TCT,GGT,GAT,GCT,CTC,AGT,ATT,TCT,AGG,CAT,GAA,AAC,GTT,AAC,AGC,TGC,AGA,AGT,TGG,AAC,TGG,TGG,TTG,GTG,GCA,GTC,CAG,GGC,ACA,CAG,CGA,GGC,TTC,TCC,CCT,GCC,ACT,CTT,TTT,TCT,GAG,GGT,TTG,GAA,GTT,TCC,TCA,GTT,GGA,GGG,AGT,GAG,AGC,TGC,TCA,TCA,AGG,ACT,TCT,CTG,TCC,GGT,TGG,AGG,TTA,ACT,CTG,TCT,CTT,GCT,CTC,TCA,TTT,CTG,CCT,GGA,CCA,AGA,CTC,TGT,GCA,CCG,AGT,CCG,CAG,ACA,TCT,TTG,CTG,CCT,CCA,AGG,GAA,GCC,GTC,CCA,CGG,TCT,GTT,TTA,GCA,AAT,GGG,GAG,ATC,CAT,CCC,CAA,ATG,TCT,GAA,CAA,GAA,ACT,TGT,CTA,ATG,GAA,AAC,GAG,CGG,GCC,CAA,ATT,AAC,TCT,AAG,GTG,TTA,GAT,GTT,TTC,AAA,GAA,CGA,GAA,GTC,TCT,TTA,CTC,TTA,AGC,ATG,TTT,TGG,TCT,TTC,TGG,TTT,CAC,TTG,ATT,AAG,ACA,TGT,AAT,AGA,AAG,CTT,ACA,TGC,TGT,AGT,CCT,GAC,TCA,GAT,CCT,GGT,CAA,AGA,AAA,GCC,CTC,TTG,GGT,TTT,ACT,CTT,TGG,CAT,AGT,GCC,TGG,AAC,GTA,GGA,GGC,ACT,CAA,ATG,CCT,TGT,ATG,AGA,GAA,TTT,TTC,TGG,CCC,ATA,CAT,TTC,AAA,ACC,AAA,TAC,TCT,CAC,AGA,AAC,AGA,TAT,GAT,GAC,AGG,TTG,AGG,GAG,CTT,TCA,TTT,TGT,CTA,AGA,GAC,TTC,CTA,TGG,CAA,CAG,AAA,AGG,TAT,CGC,CAG,AGC,CCC,TCC,TCT,TCC,ACA,GCC,TGG,CCA,CCT,AAC,AGC,CCT,CTG,GGT,TCC,GGG,GCT,GGC,CGT,CCA,GAG,CTC,CTC,AGC,TTG,CTC,TGG,CCG,GCC,GAA,CTC,CCC,TCC,AGC,TCG,GTC,TGG,AAC,CAT,CCT,GCT,GGG,CAG,CGT,CCA,GCA,CAT,CCC,TGC,TTC,GGG,CTG,CCT,GGG,CAC,CTC,GCC,TCT,CTG,CCT,CCT,GTG,CTG,CCT,CAC,CCC,CAC,CCC,TCT,ATC,TGT,AGT,GGG,AGA,GAG,ATA,GAT,TTG,ACA,GCT,GAT,AGT,GCA,TTT,TCT,CTG,ACA,AAC,ACA,CTA,CAG,CCG,TAT,CAA,TTT,TGT,GCA,TTT,CAG,TTC,CTG,TTT,TCA,TGG,AAA,CAC,ACG,GCT,GAG,AAT,GAA,AGC,CCA,AAG,CCT,CAA,TTT,CAC,AGT,GGT,CTC,CTA,ACT,ACC,TGC,TTT,CCA,TGC,AAA,CTA,GGG,AGA,TAT,GGC,CAG,GAG,AGC,CCT,GTG,TGT,TGG,GCA,GGG,TCA,CAC,TCC,AGC,ACC,CAG,ACC,ATA,GAA,CAG,GGC,CCA,TCC,TGC,TTC,ATG,AGG,GAA,ACT,GCT,CTT,CGG,GCC,TTT,AGC,TGG,ACT,ATC,TCA,TTT,CAT,TTA,TCC,CGG,GAG,TCC,GAT,ACA,GGA,GAT,TCT,GAA,GGG,CAA,ATT,ACA,CAC,TTT,TTT,TTT,TTT,TTG,AGA,GGT,CTT,GTT,CTG,TCA,CCC,AGG,CTG,GAG,TGC,AGT,GGT,GCG,ATT,TCA,GCT,CAT,AGC,AGC,CTC,CAC,CTC,CCA,GGC,TCA,AGC,TAT,CTT,CCT,ACC,TCA,GCC,TCC,CAA,GTA,GCC,GGG,ACG,ACA,GGT,GTG,CAC,CAC,CAC,GCC,TGG,CTA,ATT,TTT,GTA,TTT,TTT,TTG,AGA,TGG,AGT,CTT,GCC,ATT,TTG,CCC,AGG,CTT,GTC,TCG,AAC,TTC,TGG,GCT,CAA,GCA,ATC,CGT,CCA,CCT,CGG,CCT,CTC,AAA,GTG,CTG,GGA,TTA,GCC,ACT,GCA,CCT,GGG,CAA,CAG,TTT,ATG,TGT,GTG,TGT,GTG,TGT,GTG,TGT,GTG,TAT,ATA,TGT,GTG,TGT,ATA,TAT,ATG,TGT,GTA,TGT,ATA,TAT,GTG,TGT,GTG,TGT,GTG,TGT,GTG,TGT,GTA,AAT,CTC,CAA,GTC,CAT,CCA,ACC,GAG,AGG,CTC,CTA,CTA,GAA,GCC,AAG,AGT,CCA,CCG,GGT,GCA,CGG,GTC,TCT,GGA,GGC,CTG,TGG,CAC,TGC,GAA,GGC,TCT,AAC,AAA,GCC,AAG,GGA,AGG,GCC,ACC,TCA,CTA,GAA,GCC,AGG,CCT,GGA,GGA,AGG,GTG,AGG,GCT,GAG,GGC,CTG,GAG,GTA,AGA,CTG,CCT,GTG,GTT,TTA,GAC,CCA,GCT,CTG,CCA,CTG,ACT,AGC,TGT,GTG,GCT,GGC,CTT,CAG,CAC,ATC,TTC,ACA,CCT,CTC,TGC,ACC,TCA,GTT,TCC,ACA,TGT,GAA,GAT,ATG,AAA,GTG,ATT,CTG,AAG,GTG,ATT,GCA,AGG,TTG,ATT,GGA,ATC,CAG,CTC,TTG,AGT,TGC,AAA,GTG,TTA,TTG,GAT,GAT,ATA,ACC,ACG,ATT,AAA,AGC,AAG,AAC,AGG,TGC,AGA,GAA,GCG,TTC,GAA,GGA,GGG,GAC,CGG,GTT,GGA,AAG,GAT,CAA,ACC,TCC,AGG,ATG,CCG,AGT,CTG,GGG,CAA,TCC,ATC,TGG,GCT,GTT,TCT,GGA,AGA,CCC,CGG,GTT,GCA,GGC,CAG,GAC,ACT,GCT,GCC,CTC,CCG,TCC,TTA,ACT,CCC,CTC,TTC,ACT,CAG,TCC,TCA,CTC,ACC,TCC,CTC,TCA,CAC,ACA,CAA,CAT,CTC,CTA,GAA,TCC,CCA,CTG,CCT,GCT,TCA,CTC,TTA,CCC,GTC,TCA,TTT,GCC,TCC,CCT,GAA,CTT,CAT,CCT,CCT,GGA,GTT,CAC,GAT,CTC,ACT,CTT,CAC,TCT,TTT,CTT,CCC,CTC,GAA,GAT,TCA,GCA,CTG,CTT,ACT,TAC,ATG,TTA,AGA,TAT,TTC,AGA,ACA,GTG,AAA,TGT,TGC,TAT,TTT,CAA,AAA,CCT,ACA,AAG,GTG,GTA,TGC,AGA,GGA,AAA,GGT,ACT,TCT,TTG,TGT,TCC,CAA,AGA,AAA,CAT,CTT,TCC,AAA,ATC,CAG,CCT,ATT,GAT,TTT,ATT,TCT,TCG,GGG,GAA,CAA,GAA,TTT,TAT,CTC,GTT,GGG,CAT,TCT,ACT,CTT,GGC,AGT,TGC,TGG,AAG,AAG,GCA,CTG,GTC,GTC,CTG,GGC,TTC,ACA,GGT,AAC,ACC,TGT,CAG,GGT,GTC,TAT,GAA,GTC,AAG,GCT,GTC,GGA,ACA,GCA,AAG,TGG,GAA,GAA,GCA,GCT,GGC,TGG,CTG,ATG,AAG,GGT,TTC,TTG,GGT,GGA,CAA,GTA,GTT,GGA,GCT,ATT,TCC,TAT,TTA,CCA,AAG,AGA,GCT,AAA,GTT,CAT,AAT,TCT,ACA,GAG,AGT,TCC,ATA,ATG,AAC,CTC,AAA,TAC,CTC,TGT,TTT,TTG,AAG,GAG,TTT,CTC,ATA,TAC,AGC,ACT,AGC,CTA,TCC,TGG,GCA,GGA,TGG,GAG,ATG,ATG,CAG,TGC,CAA,TCG,GGC,TGG,ATT,TAT,ATG,GTC,CTC,AGT,GAG,GCT,GGT,CAA,GAA,CCG,AGT,AAC,TCT,CAC,AGA,GTC,ACT,GCC,ACA,GAA,GAA,ATC,TCC,CAA,GTG,GCT,GTT,TCC,CAT,TCC,CGG,GAG,GGA,CAG,GCC,TCC,TTC,GTC,ACT,CCC,GCA,GTT,CTG,AAC,TGT,GAG,GTC,AGC,CAG,GCT,GTC,CAA,GTG,CAC,TCC,CTG,AGC,CAC,TGG,CAG,ACA,CAC,TCA,GCA,GCC,AGA,GCT,AGA,CAG,GCA,GGT,GGT,AGG,AGT,CCA,GGG,CCA,CGG,CAG,GGA,TGG,AGT,GTC,GCC,CCC,CTC,GCT,GCG,ATA,CCA,GAG,CAA,CTA,AAA,CGT,GGC,CTT,GCA,CTA,AAG,CTG,CCC,CTA,GGA,TGC,ATT,CTT,TTA,AAG,TTT,TCC,ATT,TGC,AGA,CTC,TTT,TCA,ATT,CTT,ATT,TTA,TCC,TTG,TTT,CCT,TTA,GAA,AGT,CCT,TTC,AAA,AAT,ATC,TTT,AGA,GGG,TTT,TTC,CAT,ACT,ATG,TGG,CCA,TAT,ACG,GGT,CAA,AAT,GTT,TTT,CCA,GGC,TCC,AAG,CCA,GCG,TTT,CAG,AAA,ATC,TCA,CCA,AGG,TTG,TGG,AGA,AGC,AAA,GGC,CTT,TTT,GGT,TTC,CTT,GAA,TCT,CAC,TGT,TCC,CTC,TGC,AGC,AGC,ATG,CAT,GTC,TGC,CCA,CCT,CCA,GAC,ACA,CAG,GCA,CCA,TCT,GCC

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  • Search your sequence AAC,ACA,ACT,GAG,AAG,GAA,ACC,TTC,TGC,AGG,GCT,GCG,ACT,GTG,CTC,CGG,CAG,TTC,TAC,AGC,CAC,CAT,GAG,AAG,GAC,ACT,CGC,TGC,CTG,GGT,GCG,ACT,GCA,CAG,CAG,TTC,CAC,AGG,CAC,AAG,CAG,CTG,ATC,CGA,TTC,CTG,AAA,CGG,CTC,GAC,AGG,AAC,CTC,TGG,GGC,CTG,GCG,GGC,TTG
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  • Run the safety screen
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Part 5: From design to build

How would you build it? I would suggest building it from a pool of oligos synthesized on a chip due to the length and complexity of reducing the amount of error during DNA synthesis.

What does synthesis constrain? Synthesis has error rates thus the amount of errors in the synthesis would have to be reduced drastically and multiple mRNA sequences and its corresponding signalling tag might require the introduction of a plasmid per each substrate instead of a combination plasmid with all substrates in one plasmid.

Prototype vs. scale. The microfluidic automation in the HOPE factory workflow will measure the substrate concentration in the LF-OOAC and send the external signal that are detected by the vector context providing the signaling needed to enhance the substrate production. The plasmid itself will not need to be replicated, as only transcription and translation will occur upon stimulation from the automation.

Part 6: What don’t you know yet?

2–3 biggest unknowns or risks in your construct:

  • Vector context (Signal Regulatory region) is from Homo sapiens BRCA1 intron 2 regulatory region thus from a totally different gene from the genes of IL-4, IL-21 or CD40-Ligand. Thus it is unknown if it will function as detailed.
  • The signal peptide is from IL-4 gene not found in IL-21 nor CD40-Ligand. Thus it is unknown if it will function as detailed.
  • Although the simulator shows successful transcription of mRNA, I do not know if the Tfh cell simulator plasmid will successfully produce IL-4, IL-21 and CD40-Ligand when prompted to do so externally via automation.
  • Another unknown are the post translational modification issues that may arise during translation, since this is a common issue even in chassis of mammalian cells thus one must suspect the same issue in mammalian cell-free systems as well.
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AI Prompts

Kernel AI

  • How to extract mRNA sequence from the gene from GenBank

  • From repository Tfh_Michellie_Hernandez how can I extract mRNA

  • how to add parts to a sequence

  • 5’URL in public repository

  • Parts of the IL-4 gene in humans

  • which parts in a plasmid sequence are needed to stimulate the transcription and translation of mRNA for IL-4

  • How to optimize the codons

  • AAC,ACA,ACT,GAG,AAG,GAA,ACC,TTC,TGC,AGG,GCT,GCG,ACT,GTG,CTC,CGG,CAG,TTC,TAC,AGC,CAC,CAT,GAG,AAG,GAC,ACT,CGC,TGC,CTG,GGT,GCG,ACT,GCA,CAG,CAG,TTC,CAC,AGG,CAC,AAG,CAG,CTG,ATC,CGA,TTC,CTG,AAA,CGG,CTC,GAC,AGG,AAC,CTC,TGG,GGC,CTG,GCG,GGC,TTG search repositories for similar DNA sequences

OpenAI ChatGPT 5

  • Do Natural bacterial plasmids all start with 5’UTR and end with 3’UTR and a Poly A tail

  • What is the plasmid architecture in cell-free systems

  • How can I stimulate a mammalian cell-free system DNA architecture?

  • What is a Vector context backbone in the construct of a plasmid

  • What vector context can be used in cell-free system plasmid that needs to be enhanced by exterior signaling

  • What Signal peptide (secretion), tag are available in cell-free systems