Week 2 HW: DNA Design

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Part 0: Setup in Kernel.

I logged into Kernel and created a repository named “HTBAA-2026 Nana Agyei William & Mary Node”.

Part 1: Define your Expression Target.

Answer

The molecule I aim to express is the AppA phytase, a phosphate-mobilizing enzyme that releases phosphate from phytate.

Phytase AppA Protein Sequence:

MKAILIPFLSLLIPLTPQSAFAQSEPELKLESVVIVSRHGVRAPTKATQLMQDVTPDAWPTWPVKLGWLTPRGGELIAYLGHYQRQRLVADGLLAKKGCPQSGQVAIIADVDERTRKTGEAFAAGLAPDCAITVHTQADTSSPDPLFNPLKTGVCQLDNANVTDAILSRAGGSIADFTGHRQTAFRELERVLNFPQSNLCLKREKQDESCSLTQALPSELKVSADNVSLTGAVSLASMLTEIFLLQQAQGMPEPGWGRITDSHQWNTLLSLHNAQFYLLQRTPEVARSRATPLLDLIKTALTPHPPQKQAYGVTLPTSVLFIAGHDTNLANLGGALELNWTLPGQPDNTPPGGELVFERWRRLSDNSQWIQVSLVFQTLQQMRDKTPLSLNTPPGEVKLTLAGCEERNAQGMCSLAGFTQIVNEARIPACSL

Phytase AppA DNA Sequence:

ATGAAAGCGATCTTAATCCCATTTTTATCTCTTCTGATTCCGTTAACCCCGCAATCTGCAT TCGCTCAGAGTGAGCCGGAGCTGAAGCTGGAAAGTGTGGTGATTGTCAGTCGTCATGGTGTGCGTGCTCC AACCAAGGCCACGCAACTGATGCAGGATGTCACCCCAGACGCATGGCCAACCTGGCCGGTAAAACTGGGT TGGCTGACACCGCGCGGTGGTGAGCTAATCGCCTATCTCGGACATTACCAACGCCAGCGTCTGGTAGCCG ACGGATTGCTGGCGAAAAAGGGCTGCCCGCAGTCTGGTCAGGTCGCGATTATTGCTGATGTCGACGAGCG TACCCGTAAAACAGGCGAAGCCTTCGCCGCCGGGCTGGCACCTGACTGTGCAATAACCGTACATACCCAG GCAGATACGTCCAGTCCCGATCCGTTATTTAATCCTCTAAAAACTGGCGTTTGCCAACTGGATAACGCGA ACGTGACTGACGCGATCCTCAGCAGGGCAGGAGGGTCAATTGCTGACTTTACCGGGCATCGGCAAACGGC GTTTCGCGAACTGGAACGGGTGCTTAATTTTCCGCAATCAAACTTGTGCCTTAAACGTGAGAAACAGGAC GAAAGCTGTTCATTAACGCAGGCATTACCATCGGAACTCAAGGTGAGCGCCGACAATGTCTCATTAACCG GTGCGGTAAGCCTCGCATCAATGCTGACGAAGATATTTCTCCTGCAACAAGCACAGGGAATGCCGGAGCC GGGGTGGGGAAGGATCACCGATTCACACCAGTGGAACACCTTGCTAAGTTTGCATAACGCGCAATTTTAT TTGTTACAACGCACGCCAGAGGTTGCCCGCAGCCGCGCCACCCCGTTATTAGATTTGATCAAGACAGCGT TGACGCCCCATCCACCGCAAAAACAGGCGTATGGTGTGACATTACCCACTTCAGTGCTGTTTATCGCCGG ACACGATACTAATCTGGCAAATCTCGGCGGCGCACTGGAGCTCAACTGGACGCTTCCCGGTCAGCCGGAT AACACGCCGCCAGGTGGTGAACTGGTGTTTGAACGCTGGCGTCGGCTAAGCGATAACAGCCAGTGGATTC AGGTTTCGCTGGTCTTCCAGACTTTACAGCAGATGCGTGATAAAACGCCGCTGTCATTAAATACGCCGCC CGGAGAGGTGAAACTGACCCTGGCAGGATGTGAAGAGCGAAATGCGCAGGGCATGTGTTCGTTGGCAGGT TTTACGCAAATCGTGAATGAAGCACGCATACCGGCGTGCAGTTTGTAA

I obtained this sequence from NCBI : https://www.ncbi.nlm.nih.gov/nuccore/AF537219.1

10..1308 /gene=“appA” /note=“phytase; phosphatase” /codon_start=1 /transl_table=11 /product=“AppA” /protein_id=“AAN28334.1

The host would be an E.coli K-12 cell-free expression system. This is because the Pho phosphate-response system is well characterized in E.coli. and it has a native phosphate regulatory system that would serve as a sensing mechanism.

I think periplasmic targeting would be a good choice, since AppA is naturally associated with the periplasmic space in E.coli.

Part 2: Design your genetic parts.

For parts, I would use:

  • A Pho-responsive promoter such as the phoA promoter, which is a characterized E. coli phosphate-starvation-responsive promoter. (BBa_K912001)

  • BBa_B0034 as the ribosome binding site.

  • AppA phytase coding strand serving as the phosphate-mobilizing enzyme

  • BBa_B0015 as the Terminator

  • BBa_K4201013 as the backbone; this is an ampicillin backbone that confers ampicillin resistance. It uses the pUC191 vector, which is commonly used as an E.coli cloning vector.

Part 3: Assemble your construct.

My genetic construct is designed to express AppA phytase/acid phosphatase, a phosphate-modifying enzyme that helps release phosphate from organic phosphorus compounds, potentially making phosphorus available for plant uptake when availability is low.

Part 4: Optimize and quality-check.

I used the coding sequence optimization tool in Kernel to optimize the DNA sequence for AppA phytase for an E.Coli host.

  • Optimized Sequence

ATGAAAGCAATTCTGATTCCGTTTCTGAGCCTGCTGATTCCGCTGACCCCGCAGAGCGCATTTGCACAGAGCGAACCGGAACTGAAACTGGAAAGCGTTGTTATTGTTAGCCGTCATGGTGTTCGTGCACCGACCAAAGCAACCCAGCTGATGCAGGATGTTACCCCGGATGCATGGCCGACCTGGCCGGTTAAACTGGGTTGGCTGACCCCGCGTGGTGGTGAACTGATTGCATATCTGGGTCATTATCAGCGTCAGCGTCTGGTTGCAGATGGTCTGCTGGCAAAAAAAGGTTGTCCGCAGAGCGGTCAGGTTGCAATTATTGCAGATGTTGATGAACGTACCCGTAAAACCGGTGAAGCATTTGCAGCAGGTCTGGCACCGGATTGTGCAATTACCGTTCATACCCAGGCAGATACCAGCAGCCCGGATCCGCTGTTTAATCCGCTGAAAACCGGTGTTTGTCAGCTGGATAATGCAAATGTTACCGATGCAATTCTGAGCCGTGCAGGTGGTAGCATTGCAGATTTTACCGGTCATCGTCAGACCGCATTTCGTGAACTGGAACGTGTTCTGAATTTTCCGCAGAGCAATCTGTGTCTGAAACGTGAAAAACAGGATGAAAGCTGTAGCCTGACCCAGGCACTGCCGAGCGAACTGAAAGTTAGCGCAGATAATGTTAGCCTGACCGGTGCAGTTAGCCTGGCAAGCATGCTGACCAAAATTTTTCTGCTGCAGCAGGCACAGGGTATGCCGGAACCGGGTTGGGGTCGTATTACCGATAGCCATCAGTGGAATACCCTGCTGAGCCTGCATAATGCACAGTTTTATCTGCTGCAGCGTACCCCGGAAGTTGCACGTAGCCGTGCAACCCCGCTGCTGGATCTGATTAAAACCGCACTGACCCCGCATCCGCCGCAGAAACAGGCATATGGTGTTACCCTGCCGACCAGCGTTCTGTTTATTGCAGGTCATGATACCAATCTGGCAAATCTGGGTGGTGCACTGGAACTGAATTGGACCCTGCCGGGTCAGCCGGATAATACCCCGCCGGGTGGTGAACTGGTTTTTGAACGTTGGCGTCGTCTGAGCGATAATAGCCAGTGGATTCAGGTTAGCCTGGTTTTTCAGACCCTGCAGCAGATGCGTGATAAAACCCCGCTGAGCCTGAATACCCCGCCGGGTGAAGTTAAACTGACCCTGGCAGGTTGTGAAGAACGTAATGCACAGGGTATGTGTAGCCTGGCAGGTTTTACCCAGATTGTTAATGAAGCACGTATTCCGGCATGTAGCCTGTGATAA

-Searched Sequence (BLAST)

I ran a BLAST search of the AppA phytase DNA coding sequence, and it ended up being a 100% match since I obtained the sequence from NCBI.

  • Safety Screen

I ran the AppA phytase sequence through Kerenl’s biosecurity sequence scanner, and no flagged sequences were detected.

Part 5: From design to build

I would build the consturct by