https://pages.htgaa.org/2026b/zarias-arman-aelius-saadatkhah/week-02-hw-dna-design/index.htmlPart 1: Define your expression target Start from the project you proposed in Homework #1 and pin down exactly what your cells need to make. Answer in your write-up: What molecule are you expressing? A protein, an RNA, or an enzyme (or small set of enzymes) for a pathway. If your HW1 product is a material, food, or chemical, identify the protein(s) that produce it and design for those. What is the sequence? Give the protein or RNA sequence, or the specific gene, you will encode, and say where it comes from (a database accession such as UniProt or NCBI, a known part, or de novo). Cite it. What host or chassis? The living system from HW1, for example E. coli, S. cerevisiae, CHO or another mammalian line, or a cell-free system. Your host drives almost every part choice that follows. Where does the product need to go? Cytoplasm, secreted, membrane, or a compartment? This decides whether you need a signal peptide or targeting sequence. We are expressing a single engineered recombinant fusion protein acting as a trifunctional biocatalyst containing both a monomeric inorganic pyrophosphatase and an active-site-expanded terminal deoxynucleotidyl transferase. This chimeric enzyme molecule is encoded by a synthetic DNA sequence combining structural elements derived from Bacillus subtilis (ppaC architecture) and mammalian terminal deoxynucleotidyl transferase databases. The expressed protein product accumulates directly within the cytoplasm of a recombinant Escherichia coli BL21(DE3) host chassis for downstream affinity purification and immobilization. Monomeric Family II Inorganic Pyrophosphatase (mPPase) derived from Bacillus subtilis (strain 168) Family II inorganic pyrophosphatase (ppaC gene architecture). Terminal Deoxynucleotidyl Transferase (TdT Core) derived from mammalian template-independent DNA polymerase (DNTT). Rigid $\alpha$-Helical Linkers (EAAAK)₃ spacer sequence (AITEEAAAKEAAAKEAAAK) ensuring 25–30 Å spatial separation between domains. Deca-histidine sequence (HHHHHHHHHH) for immobilized metal affinity chromatography (IMAC). C-terminal SpyTag003 sequence (AHIVMVDAYKPTK) for covalent isopeptide crosslinking to solid supports. Recombinant Escherichia coli strain BL21(DE3) since lacks the outer membrane protease ompT and the cytoplasmic protease lon, significantly minimizing proteolytic degradation of recombinant fusion proteins. Also, the host strain harbors a lysogenic $\lambda$ prophage carrying the gene for T7 RNA polymerase under the control of the lacUV5 promoter allowing a tight, inducible transcription of genes cloned downstream of a T7 promoter on a companion expression vector. A final point being paired with a pET-derived expression vector (such as pET-28a) featuring a kanamycin-resistance marker and a lac operator ($lacI^q$) it will suppress basal expression during the cell-growth phase. The product will go to the cytoplasm of the recombinant E. coli BL21(DE3) host chassis. Since the engineereed 82 kDA trifunctional biocatalyst is design to accumulate as a fully solublw intracellular product, it does not require an N-terminal secretory signal peptide (such as PelB or OmpA) for periplasmic or extracellular routing. Part 2: Design your genetic parts Every construct is built from parts. Decide which parts your target needs, then create or import each one in Kernel with the correct part type. At minimum, choose the elements below for your host.Hugoen