Week 2 HW: DNA Read, Write, and Edit

Part 1: Benchling & In-silico Gel Art

1.1 Restriction Digestion Simulation in Benchling:

Restriction digestion gel image Restriction digestion gel image

1.2 DNA Gel Art Using Automation Art:

DNA Art Image DNA Art Image

Part 2: Laboratory Work on Gel Electrophoresis

Skipped due to lack of access to lab.

Part 3: DNA Design Challenge

Lorem Ipsum

3.1. Choose your protein.

Database Used: UniPort

tr|O33823|O33823_ACIFR Cytochrome c OS=Acidithiobacillus ferrooxidans OX=920 GN=cyc2 PE=4 SV=1 MVSSSVGFKKKRLIVALAAVGGMALSSSAWALPSFARQTGWSCAACHTSYPQLTPMGRMFKLLGFTTTNLQRQQKLQAKFGNSVGLLISRVSQFSIFLQASATNVGGGQAVFGSGNSNANASPNNNVQFPQQVSLFYAGEITPHIGSFLHITYSGGGSGTGGGGFSFDDSSIVWAHPWKLGTNNLLVTGVDVNNTPTAMDLWNTTPDWQAPFFSSDYSSWGHVPQPFIESSAGAGYPLAGVGVYGADIFGPNRANWLYADADVYTNGQGTQVNPVGGFTAAGPQGRLSGGAPYVRLAYQHDWGDWNWEVGTFGMWSSVYDNTLNNPLNNISKAGGPIDTFDDYDLDTQLQWLDTNDNNNVTIRAAWVNEQQQFGAGNIISSNSSGNLNFFNVNATYWYHDHYGIQGGYRNVWGSANPGLYTTTYTNSGSPDTSNEWIEASYLPWWNTRFSLRYVVYNKFNGVGSASSNNLGYGASAYNTLELLAWISY

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

Tool used: NCBI

AJ006456.2 Acidithiobacillus ferrooxidans cyc1, cyc2, coxA, coxB, coxC, coxD and rus genes and open reading frame TTGGCATGTCGATTTTTGGACCTCTAGTGATCACGGCCTATAATTAAACGGCATGGTTAACATGATAAAATAACGTTAGCACATAATTCTTTTCTTATGTTCGTTATTTACTTTATTGCATTTTACTGGATCGATATTCTGGCAACTATGCGCAAAATATTGATTATAAAAGCATTATAGTTATGACCATCGAGGCGATCGCGAGATGCATGGATGAGGTAGCCATGCATTTTAATGAGCGCATAAAAAGATGTTGCAAAGCATCGCGGTTTGTATTAAATAGAACGTGTGGGTATTGTTAACAACGCAACAACATTGGTTAAAGGTCGAGGCTAATTGGCATCGCGTTGTTGTGGTTTGGTGTTACCAGCCTGGCAGGAAGACCGGGCGCATGAGCGTATTTTGTTTATCTAATATGCCTGAAAGCGCATACCGCTATGGAGGGGGTTATGGTGTCATCGTCCGTTGGTTTTAAAAAGAAAAGGTTGATCGTAGCATTAGCAGCAGTTGGTGGAATGGCGTTATCTTCCAGTGCCTGGGCACTGCCATCCTTTGCGCGCCAGACCGGTTGGTCGTGCGCCGCCTGTCACACATCCTACCCGCAGTTGACGCCCATGGGCAGAATGTTCAAATTGCTCGGGTTCACGACCACAAACCTGCAACGGCAGCAGAAGCTCCAAGCCAAGTTCGGGAACAGCGTCGGTCTGCTCATATCCCGCGTGTCACAATTTTCTATCTTCCTGCAGGCCTCGGCGACCAATGTTGGTGGCGGGCAGGCGGTGTTTGGTTCTGGTAACTCTAATGCGAATGCTTCTCCCAACAATAATGTTCAGTTTCCACAACAGGTGAGCTTGTTCTATGCCGGTGAAATCACTCCGCATATCGGCTCGTTTCTGCATATCACCTACTCGGGCGGCGGCAGTGGTACCGGCGGCGGAGGATTTAGTTTTGACGACTCCAGCATTGTCTGGGCCCATCCATGGAAGTTGGGCACCAACAATCTTTTGGTTACGGGCGTAGACGTCAACAATACCCCGACTGCTATGGACTTGTGGAATACCACACCTGATTGGCAGGCACCATTTTTCTCCTCGGATTATTCGTCTTGGGGCCACGTACCTCAGCCATTCATTGAAAGTTCAGCGGGCGCGGGTTACCCATTAGCGGGTGTTGGTGTCTATGGGGCGGATATTTTTGGGCCAAACCGGGCAAACTGGCTGTACGCAGACGCCGATGTCTATACCAACGGTCAAGGAACCCAAGTCAACCCGGTTGGCGGTTTTACTGCAGCTGGCCCCCAGGGCAGGCTTTCAGGGGGCGCTCCTTATGTTCGTCTTGCCTATCAGCACGATTGGGGTGACTGGAACTGGGAGGTCGGCACCTTTGGCATGTGGTCCAGCGTGTACGATAACACCCTAAATAATCCTCTCAATAATATCAGCAAAGCAGGCGGCCCCATTGATACCTTCGATGATTATGATTTAGATACTCAGCTCCAATGGCTTGATACCAACGACAACAATAACGTGACGATCCGTGCCGCATGGGTAAACGAGCAGCAGCAATTTGGAGCGGGGAATATCATATCTTCGAACTCCTCCGGTAACTTAAATTTCTTCAATGTTAACGCCACCTACTGGTATCATGACCACTACGGCATTCAGGGCGGATACCGGAATGTGTGGGGGTCCGCTAACCCCGGTCTCTACACTACCACATACACTAATAGTGGTTCTCCAGATACCAGCAATGAATGGATAGAGGCTTCCTATCTGCCGTGGTGGAATACCCGCTTCTCCTTGCGATATGTCGTATACAACAAGTTCAATGGCGTTGGTTCGGCGTCGTCCAACAACCTTGGATATGGGGCGTCTGCGTATAACACCCTTGAACTGCTGGCCTGGATATCATACTAGGAGCCGATGCCATGACGACATACTTAAGCCAAGACCGGTTGCGCAATAAAGAGAACGACACGATGACCTATCAACATAGCAAGATGTATCAGTCGAGAACCTTCCTTCTGTTCAGCGCACTCTTGCTGGTGGCCGGGCAGGCGAGTGCTGCAGTCGGCAGCGCCGACGCGCCGGCACCATACCGCGTCTCCAGTGATTGCATGGTATGCCACGGGATGACGGGCCGTGACACGCTCTATCCGATCGTCCCCCGCCTGGCCGGACAGCATAAGAGTTATATGGAAGCGCAGTTGAAAGCGTATAAGGATCACTCGCGTGCGGATCAGAATGGCGAGATCTACATGTGGCCCGTGGCGCAAGCGCTGGACAGTGCGAAAATCACGGCGCTGGCAGATTACTTCAACGCCCAGAAGCCGCCGATGCAAAGCAGCGGCATCAAGCATGCCGGTGCGAAAGAAGGAAAGGCCATATTCAACCAAGGGGTTACCAACGAACAAATCCCTGCCTGTATGGAATGCCACGGATCGGATGGCCAAGGGGCGGGCCCGTTCCCCCGGCTGGCGGGCCAGCGTTACGGCTACATCATTCAGCAGTTGACCTACTTCCACAACGGCACACGGGTAAATACCCTGATGAACCAGATTGCGAAGAATATCACCGTGGCGCAGATGAAGGATGTGGCGGCTTATCTTTCATCGCTGTAAGCGTTGTAATTGGTCAATAGAAGTTTTCCTGGCAGGCTGAAGTTTATAAAAATGGGTCTGCCAGGCATTTGCACCGTCAGGTTTATGTGCTTCTCAAAGGAGGTAGAGGTATGGCAGCAAAAAAAGGTATGACTACGGTGCTTGTATCCGCCGTGATATGCGCGGGGGTAATTATAGGTGCCCTGGAGTGGGAAAAAGCGGTAGCCCTGCCCAATCCTTCCGGGCAGGTCATTAATGGGGTACATCATTATACGATCGATGAGTTCAACTATTATTATAAACCGGATCGCATGACCTGGCATGTCGGGGAAAAAGTGGAGTTGACGATTGATAACCGATCGCAATCAGCGCCCCCGATTGCGCATCAGTTCTCCATCGGCAGAACGCTGGTATCCCGGGACAATGGCTTTCCAAAATCACAGGCTATCGCCGTGGGATGGAAAGATAACTTCTTTGATGGTGTGCCGATTACCAGCGGGGGACAGACAGGGCCAGTACCGGCGTTTTCCGTCAGCCTCAACGGTGGACAAAAGTACACCTTCAGTTTTGTGGTGCCCAATAAGCCCGGAAAATGGGAATATGGGTGTTTTCTGCAGACGGGTCAACACTTCATGAATGGGATGCATGGTATTCTTGACATACTACCTGCTCAGGGAAGCTAATTTAGGGAGGGCATATGAACGCAGCAAAAGAAAACTTATGGAAAGCTTTCCGCGGCTTGGTGGTGGTCTGGATTATTGGCCTGGCGATTTTCGAAACGCTGATGGCCTGGGGTATCGGTAACTGGCCAATTTTGGGGAGTATTCAGGCGCATATTACCGCAGATGCCACCACATACCTGTTGTGGCAGGCCGTATTCATCTATGTGCTGGTCGGCGGTGCGATTGTATATAGCGCATTTCGTTTCCGCGCATCATCCATGTCAGACACCGCGGCGCCGGCTTATCAAAAACGGACCTGGGCGCCTTTCGTGGTGACCTGGCTGGTTTTGGCCATAGGCATCAACCTGGCAAATACCATTTATCCGGGTATGGTGGGTCTGGAACAACTTTGGGGTATCCAGTTAGATACGAAGAACCCATTGGTGATCGATGTTACCGCGCAACAGTGGAAGTGGACGTTCTCTTATCCTAAGCAGGGCGTAACGGATGTGTCACAACTGGTGGTTCCCGAGGGCCGCACCATATACTTCGTTCTGCGGACAAAGGATGTCATGCACGATTTTTGGGTGCCTGCCTGGGGTGAGAAAAAAGATGTGATCCCCAATGAAGTGCGGCACTTGTTTATTACACCCACCATGTTGGGGACAACCGCTACAAACCCCATGCTGCGTGTACAGTGTTCCTTGATTTGTGGCAACGGACATCCGTTGATGCGCGCTCCGGTGAAAGTGGTAACGCCAGCGGACTTCAAGGCTTGGGTGGCAAACAATAGCTTCTAGTAAAGCCAACGGAAGGCTTGCCAGCACCCAACGTTAAATGTACTAAGGAGTAAGTAATGGCAACTAACGAAATTCAGGAAAATGCGTTGAACAATACGGGAGTGGACAAGACCCCATTTGCGGCTAGCATGCTGTTTCCGCTGTTCCGTGCGACGCTTTGGGGACTAACCGGCTATTTTGCTGCGGCATGGATCACTGCTTTATTGCTCCACACGGTAATCGTAAACCCTTTACCCGCGACAGTGGGTTATGTGGCCGGCTTGGTCTGCTGGCTGATGGGCAGCGGTGTATGGGAGGGATGGATACGACGCGCATTTGGAGGAAAAGAAGCTCCAACTTACACGGGTATCGAACGTTATTTTCGCTTTGGTCCCGATTCAAAATCCGCAGCCGTACGCTACGTAATCTTAAATATACTAACGTTCTGCTTTGCCGGCATGGCCGCCATGGCGATCCGCATTGAACTGTTGACGCCAGACTCCACCAGTTGGTGGCTGTCAGAAATCCAGTACAACCAAACGTTCGGTATTCATGGATTGATGATGTTGTTGGGTGTGGTGGCCTCTGCCATCGTCGGCGGTGTTGGCTACTATCTTATCCCGTTGATGCTTGGCACGAGAAATGTAGTATTCCCAAAACTTCTTGGCCTAAGTTGGTGGCTTTTGCCACCGGCGACCTTCGCTGTTTTTATGAGTCCTACGACCGGTGGGTTTCAGACGGGATGGTGGGGATATCCGCCGTTGGCGCAAAACAGTGGTAGCGGTATTGTGTGGTATGTCCTCGGTGCCGCCACCATTCTTGTTGCGTCGCTACTTGGAGCCATCAATATCGCCGGAACCATGGTGTACATGCGCGCCAAGGGCATGAGCCTGGGTCGCGTTCCGATTTTTGTGTGGGGTTTATTTGCGGCAGCCACCACTCTCGTCGTAGAGTCGCCAGCAACCTATACCGGCGCGCTCATGGACTTATCCGACATGATCGCCGGATCGCATTTTTATACCGGTCCCACCGGCCACCCGTTAGCGTATCTCGATCAGTTCTGGTTTTTGTTCCATCCAGAGGTCTACGTTTTCATTCTGCCCGCTTTTGCCATATGGCTGGAGATTCTTCCTGCCGCGGCCAAGCGGCCGTTGTTTGCTAGGGGTTGGGCCATCGCCGGACTGGTTGGCGTTTCCATGTCGGGTGCAATGTCGGGTGTCCATCACTACTTCACTGCGGTGAGTGACGCGCGTATGCCCATATTCATGACCATAACGGAAACTGTATCCATTCCGACAGGGTTCATTTATTTGTCCGCCATCGGAACGATATGGGGTGGTCGTTTAAGAATTAATGCTGCGGTATTGCTCGTACTGATGGCGATGATGAACTTCCTGATCGGTGGGCTGACGGGCATATTCAATGCCGACGTTCCCGCCGACCTTCAGCTGCACAACACCTACTGGGTTATTGCGCATTTCCATATACGATGCTTTGGTGGAGTGATCTTTACCTGGATTGCCGCGCTATACTGGTGGTTTCCCAAGGTTACTGGACGGAAGATCAATGAATTTTGGGGAAAGTTTCACGCATGGTGGTCCTTCGTATTCTTCAATTGTACGTTCTTTCCCATGTTTATAGCTGGACTAGATGGAATGAACAGGAGAATTGCGATATATCTTCCTTACCTGCATGACATCAACCTGTTTATGTCTATTTCATCCTTTTTCTTGGGCGCAGGGTTTCTCATTCCGCTGGCCAATCTTTTATACAGTTGGCGCTATGGGCCAAAGGCCGAAGCTAACCCTTGGGGCAGCAACGGCCTGGAATGGCAAATAAAATCGCCAACACCGTATGTGCCATATCCAGCAGGAACGGAGCCAGAGGTTGTGGGCCCGAACGATAACTACGCGGCGGAAGCAAAAGACCCCTTTATTTGGGTGTCTACGCCCAGCAAGTAAATTAGAAGGAGTTGAACCATGACAGACAACAGTTATGCCAAGCTAATGGATCCGGCCTCGGAGCGTGCAAAAAGGGGTGCGTTCTTTTTCCTGATGCTTTTTGCAGCCATCATTTTTGCGATGTGGGACCTCGCGCGTTTTCTGTGGGGGCACTCGGTGCCCGCTACATTGAGCATGGGCGTGGGTGTTGCGCTGACTGTTCTGATGCTCGTCAGCCTGGTGCCGGTGATGACGGCCCGCAAAAAACTGGATCAGGGCGATGATGCCGGTATCGTGAGCAGTCTGGCAACCCTGATGGTGGTCTCGTTGGTGATGGCGGGTGGAATCGTCTACAACTGGACTACCTTAACCATCGGTAGTGGTTATGGCGGGATTTATGACATCACCAGCTTGTGGTTTCTGGTACATTTCGTGGCGGCCATCCTGGCGCTGCTGGCGAGTATCATGAAAATCACTCGCACTCCAGAGCGCGCGAAACGCGAGCGATGGGTGTCGTATAACGTGTTAACCTTCTGGGGCGGTGTGATTGTTCTATGGGTTGCATTTTTTATTGTTTTCTATATTGCGTAATGCAGTTTAGAAGATTCTCTAATGGAGTGAGGGTTAGATAATGGATATGTCACATTTATCGTTCGTTATCCCGTCTGGAGCTGATGATCCGACGTTTTTCTGGCTGACGGGGTACATTGGGTTTCCTGTGGTGTTTCTGAGTGCATACTTTTGGTGGGTATTAAAGGAGGCAAGCAAGGAAGATCGGCTGCGTATTCTAAAAAAGGGAGAAGACGGCGCATCTGGAAACGCATGATGTTCCACGGATGGTCGTGCGAGTACCGGGCGGCCATCCGGAGTTGTTTTGCGTTTTACTGTTGCGACGTCGTTATCCATGCTTCAAAGGAGGTAAATCATGAACAAGGAAGGCTGTTTAATTTCTCACGATGATCGCGATGATGGCGCATGGGATGGAAACATCGTGTTGATCATAGGATTATTGTGGGCTATTATTGCTCTGGGTGGCTATTATGTTACCCTTAGAGTGCTGTTTTGAGACAATTCCCCGGCTGGATAGGGCGATGAATACCATGTAGTAGCATATTAAAATGCCAGAGGGCCCGGTGATGGTTTTGTAGGGCGGCTGGTTCTACTCAGGTTAAACGTTAAGGAGAAGGGATAACTTATGTATACACAGAACACGATGAAAAAGAACTGGTATGTGACTGTTGGTGCGGCTGCGGCTCTGGCGGCAACGGTCGGCATGGGTACCGCGATGGCCGGCACGCTGGATTCCACATGGAAAGAGGCGACGCTTCCCCAAGTTAAGGCCATGCTGGAGAAAGATACCGGGAAAGTCAGTGGCGATACAGTTACCTACAGCGGCAAGACTGTACATGTGGTCGCGGCGGCCGTGCTCCCGGGATTTCCGTTCCCGAGCTTTGAAGTTCATGACAAAAAGAACCCGACCTTGGAGATTCCCGCAGGGGCAACCGTAGACGTGACCTTCATTAACACCAACAAGGGATTTGGTCATAGTTTTGACATCACTAAAAAAGGACCGCCTTATGCGGTTATGCCGGTGATTGACCCCATTGTCGCAGGAACTGGATTTAGCCCGGTCCCAAAAGACGGCAAGTTCGGATATACGGATTTCACCTGGCATCCGACGGCGGGTACTTACTACTACGTATGTCAGATACCGGGGCATGCCGCCACCGGTATGTTTGGTAAAATCATTGTCAAGTAAGTCCTGGATGGTTGTTGTCTGGGCAGCTGTGCTTTGCTAGTGTAGGTCCTGGTGGCCAGGGCAAATGGTTATCTTGCCCTGGCCATTGGTATTTATTATAAAATACGAATTTCATGTATTGCGTTATGCTTTGTATGATGTTATGAGTATGTTTGCATGCAACATATGATGATTGATCTAGTTTATTAAGCTATGGACCACGAAAACACGCTGCCTCGGTACATATATTAATTCATTCAGATAAAGTCCCAAACTCAGATATCCTGACG

3.3. Codon optimization (for E. coli)

Tool used: Vectorbuilder.com

AACTACACCCCGACCCCGGAAGATTGGCATGTGGATTTTTGGACCAGCAGCGATCACGGCCTGTAACTGAACGGCATGGTTAACATGATTAAATAACGCTAACACATTATCCTGTTCCTGTGCAGCCTGTTTACCCTGCTGCACTTTACCGGCAGTATCTTTTGGCAGCTGTGCGCCAAATATTAACTGTAAAAACATTACAGCTATGATCACCGCGGCGATCGCGAGATGCACGGCTAAGGCAGCCATGCGTTTTAATAAGCGCACAAAAAAATGCTGCAGAGCATTGCCGTGTGTATTAAATAAAATGTTTGGGTTCTGCTGACCACCCAACAACATTGGCTGAAAGTGGAAGCGAATTGGCACCGCGTGGTGGTCGTGTGGTGTTACCAGCCGGGCCGCAAAACCGGCCGTATGAGCGTTTTTTGCCTGAGCAACATGCCGGAGAGCGCATATCGCTATGGCGGCGGCTACGGTGTGATTGTGCGCTGGTTTTAAAAGGAAAAAGTGGACCGTAGCATTAGCAGCAGCTGGTGGAACGGCGTGATTTTTCAGTGCCTGGGCACCGCCATTCTGTGCGCGCCGGATCGCCTGGTGGTGCGCCGCCTGAGCCATATTCTGCCGGCAGTGGATGCGCATGGCCAGAACGTGCAGATTGCGCGCGTCCATGATCACAAACCGGCGACCGCGGCGGAAGCGCCGTCTCAGGTGCGTGAACAGCGCCGTTCCGCGCACATTCCGCGTGTAACCATTTTTTATCTGCCGGCCGGCCTGGGTGACCAGTGCTGGTGGCGCGCCGGCGGCGTGTGGTTTTGGTAACTGTAATGCGAATGCTTTAGTCAGCAGTGATGTAGCGTGAGCACCACCGGCGAACTGGTGCTGTGCCGTTAAAACCACAGCGCCTACCGCCTGGTGAGCGCGTATCATCTGCTGGGCCGTCGTCAATGGTATCGCCGTCGTCGCATTTAATTTTAACGCCTGCAGCATTGCCTGGGTCCGAGCATGGAAGTGGGCCATCAGCAGAGCTTTGGTTATGGCCGCCGTCGCCAACAGTATCCGGATTGCTACGGTCTGGTGGAATATCATACCTAACTGGCCGGCACCATTTTTCTGCTGGGCCTGTTCGTTCTGGGCCCGCGCACCAGCGCGATCCACTAAAAATTTAGCGGCCGCGGCCTGCCGATTAGCGGTTGCTGGTGCCTGTGGGGCGGTTATTTCTGGGCCAAACCGGGCAAACTGGCGGTGCGCCGCCGCCGCTGTCTGTATCAACGTTCGCGCAACCCGAGCCAGCCGGGCTGGCGCTTTTATTGCAGCTGGCCGCCGGGCCAGGCGTTTCGCGGCCGTAGCCTGTGTAGCAGCTGCCTGTCAGCGCGCCTGGGCTAACTGGAACTGGGCGGCCGCCATCTGTGGCACGTGGTTCAGCGCGTGCGCTAACATCCGAAATAAAGCAGCCAGTAATACCAGCAGAGCCGTCGTCCGCATTAATATCTGCGCTAACTGTAATTCCGCTATAGCGCCCCTATGGCCTAATACCAGCGTCAGCAGTAACGTGATGATCCGTGCCGCATGGGCAAACGCGCCGCGGCGATTTGGAGCGGCGAATATCACATTTTTGAACTGCTGCGTTAACTGAAATTCCTGCAGTGTTAACGCCATCTGCTGGTGAGCTAACCGCTGCGTCACAGCGGTCGCATTCCGGAATGCGTGGGCGTGCGTTAACCGCGTAGCCTGCACTACCATATTCATTAATAATGGTTTAGCCGCTATCAGCAGTAAATGGATCGCGGCTTTCTGAGCGCCGTGGTTGAATACCCGCTGCTGCTGGCCATTTGCCGCATTCAGCAGGTTCAGTGGCGCTGGTTTGGTGTTGTTCAGCAACCGTGGATTTGGGGCGTGTGTGTATAACATCCGTAAACCGCGGGCCTGGATATCATTCTTGGCGCGGATGCCATGACGACCTACCTGTCGCAGGATCGCCTGCGTAACAAAGAAAATGATACCATGACCTATCAGCATAGCAAAATGTATCAGAGCCGCACCTTTCTGCTGTTTAGCGCGCTGCTGCTGGTGGCGGGCCAGGCGAGCGCGGCCGTTGGTTCGGCAGATGCGCCGGCGCCGTACCGCGTCAGCAGTGATTGCATGGTGTGCCACGGCATGACCGGCCGTGATACGCTGTATCCTATTGTGCCGCGCCTGGCAGGCCAGCATAAAAGCTATATGGAAGCGCAGCTGAAAGCCTACAAAGATCACAGCCGCGCCGATCAGAACGGCGAAATTTATATGTGGCCGGTTGCGCAGGCCCTGGATAGCGCCAAAATCACCGCCCTGGCGGATTATTTCAATGCGCAGAAACCGCCGATGCAGAGCAGCGGTATTAAACATGCGGGCGCCAAAGAAGGCAAAGCCATTTTCAACCAGGGCGTGACCAATGAACAGATCCCGGCGTGCATGGAATGTCATGGTTCGGATGGCCAGGGTGCGGGTCCGTTTCCGCGCCTGGCCGGTCAGCGCTACGGTTACATTATTCAGCAGCTGACCTATTTTCATAACGGCACGCGCGTGAATACCCTGATGAACCAAATCGCGAAAAACATTACCGTCGCACAGATGAAGGATGTTGCGGCCTATCTGAGCAGCCTGTAAGCGCTGTAACTGGTGAACCGCAGCTTTCCGGGCCGCCTGAAATTCATTAAAATGGGCCTGCCGGGCATTTGCACGGTGCGCTTTATGTGCTTCTCGAAAGAAGTGGAAGTGTGGCAGCAGAAAAAAGTGTAACTGCGTTGCCTGTACCCGCCGTGATACGCCCGCGGCTAACTGTAAGTTCCGTGGAGCGGCAAAAAACGTTAACCGTGCCCGATTCTCCCGGGTCGTAGTCTGATGGGCTATATTATCATTCGCAGCATGAGCAGCACCATTATCATTAACCGTATTGCGTAACCCGGCATGAGCGGCAAAAAATGGAGCTAACGCCTGATCACCGATCGCAATCAGCGTCCGCGCCTGCGCATCAGCTCGCCGAGCGCGGAACGTTGGTATCCGGGCACTATGGCCTTTCAAAATCATCGCCTGTCGCCCTGGGATGGCAAAATTACCAGCCTGATGGTGTGCCGCCTGCCGGCCGGCGATCGTCAGGGCCAGTATCGCCGCTTTCCGAGCGCGTCGACCGTGGATAAAAGCACCCCTAGCGTGCTGTGGTGTCCGATTTCGCCGGAAAATGGCAACATGGGCGTGTTTTGTCGTCGCGTGAACACCAGTTAAATGGGCTGCATGGTGTTTCTGACTTACTACCTGCTGCGTGAAGCGAACCTGGGCCGCGCGTATGAACGTAGTAAACGTAAACTGATGGAAAGCTTTCCGCGTCTGGGCGGCGGCCTGGATTATTGGCCGGGCGATTTTCGTAATGCTGATGGCCTGGGCTATCGTTGACTGGCAAATTTTGGCGAATATAGCGGCGCGTACTATCGTCGTTGCCATCATATTCCGGTTGTGGCCGGCCGCATTCATCTGTGTGCCGGCCGTCGCTGCGATTGCATTTAACGCATCAGCTTCCCGCGCATTATTCATGTGCGCCATCGCGGCGCGGGTCTGAGCAAAACCGACCTGGGCGCGTTTCGCGGCGATCTGGCCGGCTTCGGCCATCGCCATCAGCCGGGCAAATACCATCTGAGCGGTTATGGCGGCAGCGGCACCACCCTGGGTTACCCGGTACGTTACGAAGAACCGATTGGTGATCGCTGCTATCGCGCGACCGTCGAAGTGGATGTGCTGCTGAGCTAAGCGGGCCGTAATGGCTGCGTGACCACTGGCGGCTCCCGTGGCCCGCACCATATCCTGCGAAGCGCGGATAAAGGCTGCCATGCGCGCTTTCTGGGCGCGTGTCTGGGCTAAGAAAAACGCTGTGATCCGCAGTAAAGCGCAGCGCTGGTTTATTACACCCACCATGTGGGTGATAACCGCTATAAACCGCATGCGGCCTGCACCGTGTTTCTGGATCTGTGGCAGCGCACCAGTGTGGATGCGCGCAGCGGCGAAAGCGGTAACGCGAGCGGCCTGCAGGGCCTGGGCGGCAAGCAGTAACTGCTGGTGAAACCGACCGAAGGCCTGCCGGCCCCGAACGTGAAATGCACCAAAGAATAAGTCATGGCGACGAACGAGATTCAGGAAAACGCCCTGAATAATACCGGTGTGGATAAAACCCCGTTCGCGGCGAGCATGCTGTTCCCGCTGTTCCGTGCGACCCTGTGGGGCCTGACCGGCTACTTCGCGGCGGCGTGGATTACCGCGCTGCTGCTGCATACCGTGATTGTGAATCCGCTGCCGGCGACCGTGGGTTATGTGGCGGGCCTGGTGTGCTGGCTGATGGGTAGCGGCGTGTGGGAAGGCTGGATTCGCCGCGCCTTTGGCGGCAAAGAAGCGCCGACCTACACCGGTATTGAACGTTACTTTCGCTTTGGCCCGGATAGCAAAAGCGCCGCCGTTCGCTACGTGATTCTGAATATCCTGACCTTTTGCTTTGCCGGCATGGCGGCGATGGCGATTCGTATTGAACTGCTGACGCCGGATAGCACCAGCTGGTGGCTGAGCGAGATCCAGTATAACCAGACCTTCGGCATTCATGGCCTGATGATGCTTCTGGGCGTTGTAGCGAGCGCCATTGTGGGCGGCGTGGGCTATTATCTGATACCGCTGATGCTGGGCACCCGTAATGTGGTCTTTCCGAAACTGCTGGGCCTGAGCTGGTGGCTGCTGCCGCCGGCAACCTTCGCGGTTTTTATGAGCCCGACCACCGGCGGCTTTCAAACTGGCTGGTGGGGCTATCCGCCGCTGGCGCAGAACAGCGGTAGCGGCATTGTGTGGTATGTACTGGGCGCGGCCACCATTCTGGTTGCGAGCCTGCTGGGCGCCATCAACATTGCCGGCACCATGGTGTACATGCGCGCGAAAGGCATGAGCCTGGGCCGCGTGCCGATTTTTGTGTGGGGTCTGTTTGCGGCAGCGACCACCCTGGTGGTTGAAAGCCCGGCCACCTATACCGGCGCGCTGATGGATCTGAGCGATATGATTGCGGGCAGCCATTTCTACACCGGCCCGACCGGTCACCCGCTGGCCTATCTGGATCAGTTCTGGTTTCTGTTTCACCCGGAAGTGTACGTGTTTATTCTGCCGGCCTTCGCGATTTGGCTGGAAATTCTGCCGGCCGCGGCCAAACGTCCGCTGTTTGCCCGCGGCTGGGCGATTGCCGGCCTGGTTGGTGTGAGCATGAGCGGCGCGATGAGCGGTGTGCATCACTACTTTACCGCGGTCAGCGATGCCCGCATGCCGATTTTTATGACCATCACCGAAACCGTGAGCATCCCGACCGGCTTTATTTACCTTAGCGCCATTGGCACCATCTGGGGCGGCCGCCTGCGCATTAACGCCGCGGTGCTGCTGGTGCTGATGGCGATGATGAACTTCCTGATCGGAGGCCTGACCGGCATTTTTAACGCGGACGTGCCGGCGGATCTGCAGCTGCATAATACCTACTGGGTGATTGCGCATTTTCATATCCGCTGTTTTGGCGGCGTGATCTTCACGTGGATCGCCGCCCTGTACTGGTGGTTTCCAAAAGTGACCGGTCGCAAAATCAATGAATTTTGGGGCAAATTTCATGCGTGGTGGAGCTTTGTTTTTTTTAATTGCACCTTCTTCCCGATGTTTATTGCCGGCCTGGATGGCATGAACCGCCGCATTGCGATTTACTTGCCGTACCTGCATGATATTAACCTGTTTATGAGCATTAGCTCTTTTTTCCTGGGCGCGGGCTTTCTGATTCCGCTGGCGAATCTGCTGTACAGCTGGCGCTATGGCCCGAAAGCGGAAGCCAACCCGTGGGGCAGCAATGGCCTGGAATGGCAGATTAAAAGCCCGACCCCGTATGTCCCGTATCCGGCGGGCACCGAACCGGAAGTGGTGGGTCCGAACGATAACTATGCGGCCGAAGCCAAAGATCCGTTTATTTGGGTGAGCACGCCGAGCAAATGAATTCGCCGCAGCTAAACCATGACCGATAATTCCTACGCCAAACTGATGGATCCGGCGAGCGAACGCGCCAAACGCGGCGCGTTTTTCTTTCTGATGCTGTTTGCCGCCATTATTTTTGCGATGTGGGATCTGGCGCGCTTTCTGTGGGGTCACAGCGTGCCGGCGACCCTGAGTATGGGCGTTGGCGTTGCGCTGACGGTGCTGATGCTGGTGAGCCTGGTACCGGTGATGACCGCGCGCAAAAAACTGGATCAGGGGGATGATGCGGGCATTGTGAGCAGCCTGGCAACCCTGATGGTGGTGAGCCTGGTGATGGCGGGCGGCATTGTTTACAACTGGACGACGCTGACCATTGGTAGTGGCTATGGCGGCATTTACGATATTACCAGCCTGTGGTTTCTGGTGCACTTCGTGGCAGCCATTCTGGCACTGCTGGCCTCGATCATGAAAATTACCCGCACCCCGGAACGTGCCAAACGCGAACGCTGGGTTAGCTATAACGTGCTGACCTTTTGGGGTGGTGTAATTGTGCTGTGGGTGGCCTTTTTTATTGTCTTCTACATTGCCTAATGTAGCCTGGAAGATAGCCTGATGGAATAAGGCCTGGATAACGGTTACGTGACCTTCATTGTGCGCTATCCGGTTTGGTCGTAATAAAGCGATGTGTTTCTGGCGGATGGCGTTCATTGGGTGAGCTGCGGCGTTAGCGAATGCATTCTGCTGGTCGGCATTAAAGGCGGCAAACAGGGTCGCTCTGCAGCGTATAGCAAAAAAGGCCGCCGTCGCCGTATTTGGAAACGTATGATGTTTCATGGCTGGAGCTGCGAATACCGTGCCGCCATTCGCAGCTGTTTTGCCTTTTACTGCTGCGATGTTGTGATTCATGCGAGCAAAGAAGTGAACCATGAACAGGGCCGCCTGTTTAATTTTAGTCGTTAAAGCCGCTAATGGCGTATGGGCTGGAAACACCGTGTGGATCATCGTATTATTGTGGGCTATTACTGCAGCGGCTGGCTGCTGTGTTACCCGTAAAGCGCGGTACTGCGCCAGTTTCCGGGCTGGATTGGTCGTTAAATCCCGTGCTCCTCGATTCTGAAATGCCAGCGCGCACGCTAATGGTTTTGCCGTGCGGCAGGTAGCACCCAGGTCAAACGCTGAGGTGAAGGTATTACCTATGTGTACACCGAACACGATGAAAAAGAACTGGTGTGCGACTGCTGGTGCGGTTGTGGTAGCGGCGGCAATGGCCGCCATGGATATCGTGATGGCCGCCATGCCGGCTTCCATATGGAACGTGGTGATGCAAGCCCGAGCTAAGGCCACGCAGGCGAACGCTATCGCGAAAGCCAGTGGCGCTATAGCTACCTGCAGCGTCAGGATTGCACCTGCGGCCGTGGTGGCCGTGCCCCGGGTATCAGCGTGCCGGAACTGTAAAGCTCGTAACAGAAAGAACCGGATCTGGGCGATAGTCGTCGCGGTAACCGCCGTCGTGATCTGCACTAACACCAGCAGGGCATTTGGAGCTAATTTTAACATCATTAAAAACGTACGGCGCTGTGCGGCTATGCCGGCGACTAACCACACTGCCGCCGCAACTGGATCTAACCGGGCCCGAAACGCCGCCAAGTGCGTATCTATGGCTTTCATCTGGCCAGCGATGGCGGCTACCTTTTGCTGCGTATGAGCGATACCGGCGCGTGTCGCCATCGCTACGTTTGGTAAAATCATTGCCAGGTTAGCCCGGGCTGGCTGCTGTCAGGCCAGCTGTGCTTTGCGAGCGTGGGCCCGGGCGGCCAGGGCAAATGGCTGAGCTGCCCGGGCCATTGGTATCTGCTGTAAAACACCAACTTCATGTATTGTGTGATGCTGTGCATGATGCTGTAAGTTTGCCTGCATGCGACCTATGATGATTAAAGCAGCCTGCTGTCGTATGGCCCGCGCAAACATGCGGCCAGCGTTCATATTCTGATTCATAGCGATAAAGTGCCGAACAGTGATATTCTGACG

3.4. You have a sequence! Now what?

The sequence is over 8 kb long. So, I would suggest the use of cosmids for cloning. The cosmid can be inserted into E. coli, and be cloned. Inside the E. coli, the sequence replicates, transcripts, and finally translates into protein. The protein from this gene is found on the outer membrane of Acidithiobacillus ferrooxidans. But, since signal peptides and chaperone proteins for the desired protein is missing in the sequence, my educated guess is that it will be found intracellularly, and must be extracted and purified for further investigations.

Alternatively, the cell free method PURE (Protein synthesis Using Recombinant Elements) can also be used because of its faster turn around times. The DNA template strand is incubated in the presence of specific enzymes and cell extracts. The protein obtained must be purified through affinity chormatography.

4 DNA Synthesis Order

4.1 Creating accounts on Twist and Benchling:

Done.

4.2 Parts:

Lorem Ipsum

4.2.2: Promoter

TEF1

4.2.3: RBS

RBS1

4.2.4: Start Codon

ATG

4.2.5: Codon Sequence

Temporin 1 CE A.

atgttcaccttgaagaaatccctgttgctccttttcttccttgggaccatcaacttatctctctgtgaggaagagagagacgccgatgaggaagaaagaagagatgatcccgaagaaagggctgttgaagtggaaaaacgatttgtagat ttgaaaaagattgcaaatattatcaattctatatttggaaaataaccccaaaattgtaaaacttttgaaatgaaattggaaatcatctgatgtggaatatcatttagctaaatgcatatcagatgtcttacaaaaaataaagatatcacatgcaaaaaaaaaaaa

4.2.6: 7X His Tag

CATCACCATCACCATCATCAC

4.2.7: Stop Codon

TGA

4.2.8: Terminator

PGK1

4.3 Completed Plasmid

Plasmid Plasmid

Part 5: DNA Read/Write/Edit

5.1 DNA Read

a. What DNA would you want to sequence (e.g., read) and why?
I would love to sequence the antifreeze protein gene from Leucosporidium sp..The protein has a lot of applications in food technology, and medicine, and I would love to produce it commercially.

I b. What technology or technologies would you use to perform sequencing on your DNA and why?
I would use SMRT (Single Molecule Real Time) sequencing technology from PacBio. It can generate long reads (10-25 kb) with Q40+ accuracy. It is also best used for de novo genome assembly.

c. Is your method first-, second-, or third-generation (or other)?
It is a third generation sequencing method.

d. What is your input? How do you prepare your input (fragmentation, adapter ligation, PCR)?
i) DNA has to be extracted and must be purified to make it free from proteins and RNA. Long and unbroken molecules are considered to be ideal. Freshly exracted DNA is preferred over stored one.
ii) The DNA is enzymatically sheared into 10-25 kb long fragments
iii) SMRTbell library format is preferred for the preparation of library, where hairpin adapters are ligated to both 5’ and 3’ ends to create a circular template of DNA fragment. iv) Sequencing primers and the appropriate polymerases are added to the buffer containing the DNA. v) It is then loaded on to SMRT cell, that contain zero-mode waveguides. Each ZMW captures a single DNA molecule for sequencing.

e. List the essential steps.
Answered above.

f. How does your chosen sequencing technology decode bases (base calling)?
Each nucleotide contains a unique fluorescent labels, which get excited with a laser whenever a new base is added. The instrument then records the color and timing of each flash, which corresponds to the base that has been added.

g. What is the output?
HiFi reads, usually 10-25 kb long are obtained as output.

5.2 DNA Write

a. What DNA would you want to synthesize (e.g., write) and why?
I want to synthesize the Temporin 1 CE A gene found in frogs. It is a small peptide antimicrobial, and can be used to combat antibiotic-resistant bacteria.

b. What technology or technologies would you use to perform DNA synthesis and why?
Phosphoaramidite method, followed by Gibson assembly can be used to synthesize it.

c. Essential steps of chosen synthesis methods
See Homeowrk 1 for the steps of phosphoaramidite synthesis.
Steps of Gibson Assembly:

  1. Mix the pure, synthesized fragments into the reacton mix containing 5’ exonuclease, DNA polymerase, and DNA ligase. It is essential to ensure that the synthesized fragments have 15-30 bp overalaps to prevent random ligations.
  1. Incubate the samples at 50 degree celcius. This ensures that only cannonical base pairing (A=T and G≡C) occurs and non canonical bonds are prevented due to their instability at this temperature.
  2. exonuclease cleaves the -OH group, polymerase adds the nucleotides, and ligase binds the sequneces together.

d. Limitations (speed, accuracy, scalability)
Phosphoaramidite method is limited by its inability to synthesise fragments longer than 200 bp, poor yields for longer fragments, and relatively higher cost per synthesized base pairs. Gibson assembly is limited by its dependency on overlaping fragments that need to be precise. The assembled sequences must be sequenced again to make sure that it is accurate and misjoins and mutations have not occured.

5.3 DNA Edit

a. What DNA would you want to edit and why?
I would edit RSL4 gene in plants, since its overexpression increases root hair length. Longer root hairs allow the plant to uptake more nutrients.

b. What technology or technologies would you use to perform DNA edits and why?
I would use CRISPR/Cas9 because it allows precise, targeted edits and can be adapted for either gene activation or promoter replacement to drive RSL4 overexpression.

c. How does your technology edit DNA?
CRISPR/Cas9 uses a guide RNA to direct the Cas9 nuclease to a specific DNA sequence, where it introduces a double‑strand break. Repair pathways or engineered activators then modify or enhance gene expression.

d. Essential steps

  1. Design guide RNAs targeting the RSL4 promoter or coding region
  2. Clone them into a CRISPR vector
  3. Deliver the construct into plant cells
  4. Select transformed cells and regenerate whole plants

e. Preparation needed (design steps)
Identify target sites in the RSL4 promoter, ensure PAM sequences are present, and design guide RNAs with minimal off‑target potential. Choose a strong promoter or CRISPR activation system to boost expression.

f. Inputs (DNA template, enzymes, plasmids, primers, guides, cells)
Inputs include the RSL4 gene sequence, Cas9 enzyme, guide RNAs, plasmid vectors with promoters, plant cells for transformation, and primers for verification PCR.

g. Limitations (efficiency, precision)
CRISPR editing efficiency can vary across plant species, and off‑target effects may occur. Regeneration of edited plants is time‑consuming, and overexpression may cause unintended growth trade‑offs.