Week 2 DNA READ, WRITE, & EDIT

PART 1

BENCHLING RESTRICTION ENZYME SIMULATIONS ON LAMBDA DNA

GEL ART DESIGNS

DNA Design

3.1

my chosen protein for this was fibroin, which is the protein that constitutes the core silk ﬁber extracted from Bombyx mori(domestic silkworm) and consists of three subunits,ﬁbroin H chain, ﬁbroin L chain, and ﬁbrohexamerin. specifically the Fib-l - silk fibroin light chain

3.2 Reverse Translate:

786 base sequence reverse translated through https://www.bioinformatics.org/sms2/rev_trans.html

atgaaaccgatttttctggtgctgctggtggcgaccagcgcgtatgcggcgccgagcgtgaccattaaccagtatagcgataacgaaattccgcgcgatattgatgatggcaaagcgagcagcgtgattagccgcgcgtgggattatgtggatgataccgataaaagcattgcgattctgaacgtgcaggaaattctgaaagatatggcgagccagggcgattatgcgagccaggcgagcgcggtggcgcagaccgcgggcattattgcgcatctgagcgcgggcattccgggcgatgcgtgcgcggcggcgaacgtgattaacagctataccgatggcgtgcgcagcggcaactttgcgggctttcgccagagcctgggcccgttttttggccatgtgggccagaacctgaacctgattaaccagctggtgattaacccgggccagctgcgctatagcgtgggcccggcgctgggctgcgcgggcggcggccgcatttatgattttgaagcggcgtgggatgcgattctggcgagcagcgatagcagctttctgaacgaagaatattgcattgtgaaacgcctgtataacagccgcaacagccagagcaacaacattgcggcgtatattaccgcgcatctgctgccgccggtggcgcaggtgtttcatcagagcgcgggcagcattaccgatctgctgcgcggcgtgggcaacggcaacgatgcgaccggcctggtggcgaacgcgcagcgctatattgcgcaggcggcgagccaggtgcatgtg

3.3. Codon optimization

ATGAAACCGATTTTTCTGGTGCTGCTGGTGGCGACCAGCGCGTATGCGGCGCCGAGCGTGACCATTAACCAGTATAGCGATAACGAAATTCCGCGCGATATTGATGATGGCAAAGCGAGCAGCGTGATTAGCCGCGCGTGGGATTATGTGGATGATACCGATAAAAGCATTGCGATTCTGAACGTGCAGGAAATTCTGAAAGATATGGCGAGCCAGGGCGATTATGCGAGCCAGGCGAGCGCGGTGGCGCAGACCGCGGGCATTATTGCGCATCTGAGCGCGGGCATTCCGGGCGATGCGTGCGCGGCGGCGAACGTGATTAACAGCTATACCGATGGCGTGCGCAGCGGCAACTTTGCGGGCTTTCGCCAGAGCCTGGGCCCGTTTTTTGGCCATGTGGGCCAGAACCTGAACCTGATTAACCAGCTGGTGATTAACCCGGGCCAGCTGCGCTATAGCGTGGGCCCGGCGCTGGGCTGCGCGGGCGGCGGCCGCATTTATGATTTTGAAGCGGCGTGGGATGCGATTCTGGCGAGCAGCGATAGCAGCTTTCTGAACGAAGAATATTGCATTGTGAAACGCCTGTATAACAGCCGCAACAGCCAGAGCAACAACATTGCGGCGTATATTACCGCGCATCTGCTGCCGCCGGTGGCGCAGGTGTTTCATCAGAGCGCGGGCAGCATTACCGATCTGCTGCGCGGCGTGGGCAACGGCAACGATGCGACCGGCCTGGTGGCGAACGCGCAGCGCTATATTGCGCAGGCGGCGAGCCAGGTGCATGTG

due to the size and highly repetetive nature of the amino acid sequences, pichia pastoris would be used to express the protein.

3.4 further production factors

for my current project i could follow the traditional route of introducing the host dna into p.pastoris through a plasmid - this could potentially be phase 1 of the project.

however i would like to work towrds using the b.mori itself through crispr/cas9, as a reactor for the protein. this could be done via injecting existing silkworm biroin with edited elements allowing the worm to spin silk within its natural condiitons.

TWIST ORDER

my twist file keeps on crashing im in the process of trouble shooting

5.1 DNA Read

What DNA would you want to sequence and why?

The genome of Bombyx mori (the domestic silkworm), specifically the fibroin heavy chain (fib-H), fibroin light chain (fib-L), and sericin genes — the core genes responsible for silk production.

What sequencing technology would you use?

A combination of Illumina short-read (2nd generation) for accuracy and Oxford Nanopore (3rd generation) for long reads to resolve the highly repetitive silk gene sequences. Extract genomic DNA from silk gland tissue For Illumina: fragment DNA (~300–500 bp), perform end-repair, ligate adapters, PCR amplify library For Nanopore: minimal fragmentation preferred (long fragments >10 kb); ligate motor proteins + sequencing adapters; no PCR needed

DNA write

What DNA would you want to synthesise and why?

synthesise a modified fibroin heavy chain coding sequence

DNA Edit

What DNA would you want to edit and why?

edit the fibroin heavy chain gene with an engineered chimeric silk sequence. This way the transgene is expressed under the native fib-H promoter.

What technology would you use?

CRISPR-Cas9