Week 1 HW: Principles and Practices

Part 1: Benchling & In-silico Gel Art

Gel lane 1: LAMCG - MboI Gel lane 2: LAMCG - MwoI Gel lane 3: LAMCG - PshAI Gel lane 4: LAMCG - MwoI Gel lane 5: LAMCG - MboI image

Part 3: DNA Design Challenge

I am very intersted for Interleukin-6 (IL-6), an protein indicating early inflammation & immune activation.

Here is the protein sequence:

CAA31312.1 IL-6 receptor precursor (AA -19 to 449) [Homo sapiens] MLAVGCALLAALLAAPGAALAPRRCPAQEVARGVLTSLPGDSVTLTCPGVEPEDNATVHWVLRKPAAGSH PSRWAGMGRRLLLRSVQLHDSGNYSCYRAGRPAGTVHLLVDVPPEEPQLSCFRKSPLSNVVCEWGPRSTP SLTTKAVLLVRKFQNSPAEDFQEPCQYSQESQKFSCQLAVPEGDSSFYIVSMCVASSVGSKFSKTQTFQG CGILQPDPPANITVTAVARNPRWLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTTWMVKDLQHHCVIH DAWSGLRHVVQLRAQEEFGQGEWSEWSPEAMGTPWTESRSPPAENEVSTPMQALTTNKDDDNILFRDSAN ATSLPVQDSSSVPLPTFLVAGGSLAFGTLLCIAIVLRFKKTWKLRALKEGKTSMHPPYSLGQLVPERPRP TPVLVPLISPPVSPSSLGSDNTSSHNRPDARDPRSPYDISNTDYFFPR

Here is the reverse translated DNA (nucleotide) sequence in both “Most likely codons” option and “Consensus codons” option:

reverse translation of CAA31312.1 IL-6 receptor precursor (AA -19 to 449) [Homo sapiens] to a 1404 base sequence of most likely codons. atgctggcggtgggctgcgcgctgctggcggcgctgctggcggcgccgggcgcggcgctg gcgccgcgccgctgcccggcgcaggaagtggcgcgcggcgtgctgaccagcctgccgggc gatagcgtgaccctgacctgcccgggcgtggaaccggaagataacgcgaccgtgcattgg gtgctgcgcaaaccggcggcgggcagccatccgagccgctgggcgggcatgggccgccgc ctgctgctgcgcagcgtgcagctgcatgatagcggcaactatagctgctatcgcgcgggc cgcccggcgggcaccgtgcatctgctggtggatgtgccgccggaagaaccgcagctgagc tgctttcgcaaaagcccgctgagcaacgtggtgtgcgaatggggcccgcgcagcaccccg agcctgaccaccaaagcggtgctgctggtgcgcaaatttcagaacagcccggcggaagat tttcaggaaccgtgccagtatagccaggaaagccagaaatttagctgccagctggcggtg ccggaaggcgatagcagcttttatattgtgagcatgtgcgtggcgagcagcgtgggcagc aaatttagcaaaacccagacctttcagggctgcggcattctgcagccggatccgccggcg aacattaccgtgaccgcggtggcgcgcaacccgcgctggctgagcgtgacctggcaggat ccgcatagctggaacagcagcttttatcgcctgcgctttgaactgcgctatcgcgcggaa cgcagcaaaacctttaccacctggatggtgaaagatctgcagcatcattgcgtgattcat gatgcgtggagcggcctgcgccatgtggtgcagctgcgcgcgcaggaagaatttggccag ggcgaatggagcgaatggagcccggaagcgatgggcaccccgtggaccgaaagccgcagc ccgccggcggaaaacgaagtgagcaccccgatgcaggcgctgaccaccaacaaagatgat gataacattctgtttcgcgatagcgcgaacgcgaccagcctgccggtgcaggatagcagc agcgtgccgctgccgacctttctggtggcgggcggcagcctggcgtttggcaccctgctg tgcattgcgattgtgctgcgctttaaaaaaacctggaaactgcgcgcgctgaaagaaggc aaaaccagcatgcatccgccgtatagcctgggccagctggtgccggaacgcccgcgcccg accccggtgctggtgccgctgattagcccgccggtgagcccgagcagcctgggcagcgat aacaccagcagccataaccgcccggatgcgcgcgatccgcgcagcccgtatgatattagc aacaccgattatttttttccgcgc

reverse translation of CAA31312.1 IL-6 receptor precursor (AA -19 to 449) [Homo sapiens] to a 1404 base sequence of consensus codons. atgytngcngtnggntgygcnytnytngcngcnytnytngcngcnccnggngcngcnytn gcnccnmgnmgntgyccngcncargargtngcnmgnggngtnytnacnwsnytnccnggn gaywsngtnacnytnacntgyccnggngtngarccngargayaaygcnacngtncaytgg gtnytnmgnaarccngcngcnggnwsncayccnwsnmgntgggcnggnatgggnmgnmgn ytnytnytnmgnwsngtncarytncaygaywsnggnaaytaywsntgytaymgngcnggn mgnccngcnggnacngtncayytnytngtngaygtnccnccngargarccncarytnwsn tgyttymgnaarwsnccnytnwsnaaygtngtntgygartggggnccnmgnwsnacnccn wsnytnacnacnaargcngtnytnytngtnmgnaarttycaraaywsnccngcngargay ttycargarccntgycartaywsncargarwsncaraarttywsntgycarytngcngtn ccngarggngaywsnwsnttytayathgtnwsnatgtgygtngcnwsnwsngtnggnwsn aarttywsnaaracncaracnttycarggntgyggnathytncarccngayccnccngcn aayathacngtnacngcngtngcnmgnaayccnmgntggytnwsngtnacntggcargay ccncaywsntggaaywsnwsnttytaymgnytnmgnttygarytnmgntaymgngcngar mgnwsnaaracnttyacnacntggatggtnaargayytncarcaycaytgygtnathcay gaygcntggwsnggnytnmgncaygtngtncarytnmgngcncargargarttyggncar ggngartggwsngartggwsnccngargcnatgggnacnccntggacngarwsnmgnwsn ccnccngcngaraaygargtnwsnacnccnatgcargcnytnacnacnaayaargaygay gayaayathytnttymgngaywsngcnaaygcnacnwsnytnccngtncargaywsnwsn wsngtnccnytnccnacnttyytngtngcnggnggnwsnytngcnttyggnacnytnytn tgyathgcnathgtnytnmgnttyaaraaracntggaarytnmgngcnytnaargarggn aaracnwsnatgcayccnccntaywsnytnggncarytngtnccngarmgnccnmgnccn acnccngtnytngtnccnytnathwsnccnccngtnwsnccnwsnwsnytnggnwsngay aayacnwsnwsncayaaymgnccngaygcnmgngayccnmgnwsnccntaygayathwsn aayacngaytayttyttyccnmgn

Here is the codon optimized sequence:

  1. based on reverse translation of most likely codons. ATGCTGGCCGTCGGCTGTGCACTGCTGGCCGCCCTGCTGGCAGCCCCCGGCGCCGCTCTGGCTCCCAGAAGGTGTCCCGCTCAGGAGGTGGCCAGAGGCGTGCTGACCTCCCTGCCAGGCGATTCCGTTACCCTGACCTGTCCAGGCGTGGAGCCCGAAGATAACGCCACCGTGCACTGGGTGCTGAGGAAACCCGCCGCCGGCTCCCATCCAAGCAGATGGGCAGGCATGGGGAGGAGACTGCTTCTGAGATCTGTCCAGCTGCACGACAGTGGGAACTATTCCTGTTACAGGGCTGGGAGGCCTGCCGGAACAGTGCATCTCCTGGTTGATGTGCCCCCAGAGGAGCCGCAGCTGTCTTGCTTCAGGAAGAGCCCCCTGAGCAATGTGGTGTGCGAATGGGGCCCCCGGAGTACCCCCAGCCTGACAACCAAAGCCGTGCTGCTGGTGAGGAAGTTTCAGAACAGCCCCGCTGAAGACTTTCAGGAGCCCTGCCAGTATAGCCAGGAGTCCCAGAAGTTTTCCTGCCAGCTGGCCGTGCCCGAGGGAGATAGCAGCTTCTACATCGTCTCCATGTGCGTGGCCTCTTCCGTGGGAAGTAAGTTTTCTAAGACCCAGACCTTCCAGGGCTGCGGCATCCTGCAGCCAGATCCTCCCGCCAATATCACAGTGACAGCCGTGGCCAGAAACCCCAGATGGCTGAGCGTGACCTGGCAGGACCCACACTCTTGGAATAGCTCTTTCTATAGGCTGAGGTTCGAGCTGAGATACAGAGCTGAGCGGTCCAAGACATTCACCACTTGGATGGTGAAGGACCTGCAGCATCACTGCGTGATTCACGATGCTTGGAGCGGCCTGAGGCATGTGGTGCAGCTGAGGGCCCAGGAAGAATTTGGGCAGGGGGAATGGAGTGAATGGTCCCCAGAGGCCATGGGGACACCCTGGACCGAGTCCAGGAGCCCACCAGCAGAAAATGAGGTGAGCACTCCCATGCAGGCTCTGACCACCAACAAAGACGATGATAACATTCTCTTCAGAGACTCTGCCAACGCCACCTCCCTGCCCGTGCAGGACAGCAGCTCCGTCCCTCTGCCAACCTTTCTGGTGGCCGGAGGCTCTCTGGCCTTTGGGACCCTGCTGTGTATCGCAATCGTGCTGAGGTTTAAGAAAACCTGGAAGCTGCGGGCCCTGAAGGAGGGAAAGACAAGCATGCACCCACCTTACTCCCTGGGACAGCTGGTGCCAGAGAGGCCCAGGCCTACACCTGTGCTGGTGCCCCTGATCTCCCCTCCTGTGTCTCCTTCCTCTCTGGGTTCTGACAATACAAGTAGCCACAACCGGCCAGACGCCAGGGACCCCCGAAGCCCCTATGATATCTCTAATACCGATTACTTCTTTCCAAGA

  2. based on reverse translation of consensus codons. (not working, as the sequence length is not a multiple of 3)

Codon optimization is a necessary step because of codon bias. Although multiple codons can encode the same amino acid, different organisms prefer certain codons over others. This preference affects: 1/ Translation efficiency, codons that match abundant tRNAs in the host organism are translated faster. 2/ Protein expression levels, Using preferred codons increases protein yield. 3/ mRNA stability, some codons help stabilize the mRNA, reducing degradation. 4/ Reduced secondary structure, optimized sequences minimize hairpins or repetitive sequences that hinder transcription/translation. 5/ Accurate protein folding, efficient translation reduces ribosome stalling, improving proper folding. Without codon optimization, a gene might produce very little protein in the chosen host.

Part 5

(i) What DNA would you want to sequence and why?

Chosen DNA

I would like to sequence DNAs sampled from human bodily fluids, household sinks, wastewater streams.

Why this DNA?

I am interested how ubiqutous DNA samples can construct a genetic reality to how we preserve healthcare and preventative monitoring.

(ii) Sequencing technology choice

Technology selected

[Name the sequencing technology (e.g., Sanger sequencing, Illumina sequencing, Oxford Nanopore, PacBio SMRT, etc.)]

Why this technology?

[Explain reasons such as read length, accuracy, cost, throughput, real-time analysis, portability, etc.]

Generation of sequencing

  • [First-generation / Second-generation / Third-generation]
  • Explain why it belongs to this category.

Input and sample preparation

Describe the required input and preparation steps, for example:

  1. DNA extraction
  2. Fragmentation (if required)
  3. Adapter ligation
  4. PCR amplification (if required)
  5. Library preparation
  6. Loading onto sequencing platform

Essential sequencing steps and base calling

[Explain how the technology reads DNA bases — e.g., fluorescence detection, nanopore electrical signal changes, synthesis-based imaging, etc.]

Output

[Describe output format — e.g., short reads, long reads, FASTQ files, sequence quality scores, aligned genomes.]

5.2 DNA Write

(i) What DNA would you want to synthesize and why?

Chosen DNA construct

[Describe the DNA you wish to synthesize — gene, operon, genetic circuit, DNA origami design, therapeutic construct, etc.]

Why synthesize this DNA?

[Explain the intended application — medicine, sensors, biomaterials, art, storage, etc.]

Proposed sequence (if available)

[Insert DNA sequence here, if known]