Week 2 HW: DNA Design

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Part 0: Set up in Kernel Repository: Cesar_Alcantara-SeedPods

Part 1: Define your expression target Molecules expressed: Ectoine biosynthesis enzymes (EctA, EctB, EctC) Sequences: The ectABC operon sequence derived from Leptospirillum ferriphilum YR01, retrieved from NCBI

  • EctA:
    1 gtggacagca aaaatatttc tccggcctgt ctggatttcc agaccccgac ccgcacagaa 61 gcggaacgtt tcttccggtt ccggacgccg acagaccttg atggccggtc tgttcatcgc 121 ctgatctcgc gctgtccgcc gctggatgcc aactccgtct atgccaatct cctccagtgt 181 ctccattttt ccggaacatc catccttgca gaaacaggag acggacagct ggcagcattc 241 atctccggat atgtgcttcc ggaaaacccg gacacgcttt tcatctggca ggtcgcggtc 301 gatcccgttc accgggggaa gggagtcgcc ctgtccatgc ttgacgcgct cctcgaccgg 361 acgattcctg cgggcgtccg ctatctggaa accacgatct ccccggaaaa cggtccctcc 421 cggaccttgt tccagaagct tttctcccgg aggaaagcgc cgttttccaa acgtcctctc 481 ttcggcagag agacacattt cgggggaggg cacgacgacg aggtcctgta tcgcggaggg 541 cccttttccg ccgtcggccc atga

  • EctB: 1 atgaagattt ttgaagaaaa cgaatccgtc gtccgcagtt attgtcggtc ttttcccgcc 61 attttccggg aagcccgcgg cgccgaactg atttcgacgg aaggggacca ctatctcgac 121 tttctcgcgg gagcgggcac cctgaactac ggacacaacc accctgtcct caagaaagcc 181 ctgatcaact acatccagga agacgggatt acccacagcc tggacctcta tacaacggcc 241 aaggagcgat ttatcgagac gttcaaccgg ttgatcctga atccccgcgg tatgggaaat 301 taccgcatgc agtttacggg accgacggga gccaacgcag tggaagccgc actcaagctt 361 gcacgaaaga tcacaggacg aaccaacatc ctgagcttta ccaacggctt tcatggctgc 421 tccatcgggg cccttgccgc gacaggaaac cgtcatcacc gtggcggggc aggcgttccg 481 ctgtctcatg tcagtcgcat gccctatgcc aactacttcg gcgaacaggt caataccatc 541 gccatgatgg acaagatgct caacgaccct tcgagcggcg tcgacaaacc ggccgcagcc 601 atcgtcgagg ttgtccaggg agagggggga ctgaataccg cttcccccga ctggatgcga 661 aagcttgaaa aactctgtcg aaagcacggc atgcttctga tcctggacga tatccaggcc 721 ggatgcggcc ggacaggtca ttttttcagt tttgaggaca tggggatccg tcctgacatt 781 gtcaccctgt ccaagtccct gagcggattc ggactgccgc ttgcgatcgt gctgatgaaa 841 gaggatctcg accaatggaa agccggcgag cacaacggca cttttcgcgg gaacaatcac 901 gctttcgtca cggccaccgt ggctcttgag catttctgga gcgatgatgc cttttcccgg 961 agcatccggg gaaaaagccg gcttctttcc gaacggttaa accgtatcgt ccttcgtcac 1021 ggcccccatt ccctcaaggt gaaagggcgg gggatgatgc agggcattgc ctgtccggac 1081 ggggaaacgg ccgaagccat ctgcacgcgc gcctttgata acggactgat tcttgaaacg 1141 tgcggctcca acgccgaagt cgtgaaatgc ctcagtccgc tgaccatcac caaagaacag 1201 atcgaccggg ccatggatat cctcgaagag gcgttttccg ccgtcctctc cgaacccgtt 1261 tccagccggt cttcctga

  • EctC: 1 atgattgtcc gaacattgga agaagccgaa caaagcgagc gaagggtggt gacgcctacc 61 tgggaaagca cacgcctgct cctgaaaaat gacaggatgg gcttttcatt ccatatcacg 121 accatctacg ccaataccga aacccacatc tgctaccaga accacctgga agccgtttat 181 tgcatcagcg gagaaggaga ggtcgagaca ctggccgacg gcgtggtcca taaaattcgg 241 cctggaacgc tctatgttct cgatcggcac gacgaccacc tgttaagagg catttcggag 301 atgaagctcg cctgtgtctt caaccctccc ctgagcggaa aagaagtgca tgacgaaaag 361 ggggtctacc cgctggaggc cgaacccatc tcctga

Chassis: Halomonas bluephagenesis

Destination of products: Cytoplasm

Part 2: Design your genetic parts Promoter: From Leptospirillum ferriphilum YR01 ectA AAAAATCCTTCAAAAACAAAGGGCATTTTTGTCATTGGAAACAAAAAAATTGGATTTCAGGTTTGACATT CCGGCGTTCACTTGCAAAACTAGAAAACATGCACAAACGGTTTGAGGGGTTTTAAACGTTGCCCTGCCGG CGTGTCGCTGGCAGGGGGGGGAAGCTGTTTTTGAAACGAGAGGGAGGCATGACGTTCGACGCGTCATGCA GGAAGGAGGAGAAGAAGCCCGAAAAACAACACGATTTCTGACTTTCCGGAAGAGAATTTCCGGACAGAAA AAGGTCATCTCTAAACCGCCCGTTTTACCCGACATCGGACCCCGAAAAAGTTCACCCCCGCAACCCCTGA ACAAAGGCACCACAACAAGAGTTTCTCTCCGGACCGGCCAGATGGCCAATTGTTTTTTCTCCGGTGACGC GGTTCTCATAAATGCATGTTGCCCGTTCAGCATCCTGACCGGACGCTCGTTTTATGACATATGCCAAAGG AGGGACGCCC

RBS: RBS (Elowitz 1999) - B0034 AAAGAGGAGAAA

CDS: EctABC

Terminator: Double terminator - B0015 CCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGT GAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA

Vector context:

Part 3: Assemble your construct cover image cover image

Part 4: Optimize and quality-check a short paragraph per tool covering what you ran, what it returned, and what you concluded. Screenshots welcome.

Part 5: From design to build How would you build it? What does synthesis constrain? Synthesis has error rates and struggles with certain sequences. Does your design contain anything hard to synthesize? How would error correction or fragment-based assembly change your approach? Prototype vs. scale. Mirroring HW1: the way you’d build one construct to test your idea may differ from how you’d produce DNA at the scale your product needs. Briefly describe both if they differ.

Part 6: What don’t you know yet?