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Mulai mengerjakan HW3 — October 5, 2026
Halaman ini berisi pekerjaan nyata untuk Bagian A: jawaban A1–A3 dan tabel konsentrasi dari dua artikel. Jawaban Inggris merupakan draf yang telah diperiksa terhadap sumber; Naufal masih perlu membacanya dan menjelaskan alasannya saat recitation. Pembaruan 5 Oktober 2026: tiga komposisi B telah tersimpan di Coopetition; bukti dan ID ada pada HW3 — Tiga Desain: Komposisi, Alasan, dan Bukti Pengajuan. Hasil eksperimen belum tersedia.
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Kembali ke HTBAA 2026 — Jurnal Homework 3: Desain Reaksi Cell-Free.
Kelas menyediakan mesin pembuat protein dalam lysate, instruksi DNA, dan base buffer. Tugas kita memilih campuran bahan pelengkap supaya mesin tersebut menghasilkan protein yang berfungsi dengan biaya yang lebih baik. Untuk pengumpulan awal, tugasnya adalah membandingkan resep, merancang tiga komposisi, mempublikasikannya di Coopetition, dan mendokumentasikan alasannya. Eksperimen menjadi tahap berikutnya menurut timeline kelas.
Tiga targetnya adalah sfGFP sebagai reporter fluorescence, TfCut2/PETase sebagai enzim, dan Reteplase sebagai protein yang menuntut pelipatan disulfida. Protein yang berbeda dapat membutuhkan kondisi campuran yang berbeda.
Baca jawaban Inggris di bawah satu bagian pada satu waktu. Setelah membaca A1, coba jelaskan dengan satu kalimat mengapa nilai magnesium tidak bisa dipilih terpisah dari sumber energi.
Potassium and magnesium glutamate help establish the ionic environment in which cell-free transcription and translation operate. Their useful concentrations depend on the whole formulation, including the lysate, its carried-over salts, nucleotides, energy substrates, and metabolites that accumulate during the reaction. A concentration optimized for one system should therefore be re-evaluated when those conditions change. Jewett and Swartz explicitly reoptimized magnesium when replacing PEP with pyruvate because PEP has a stronger affinity for magnesium and the expected phosphate accumulation also changes. Jewett & Swartz, 2004, PDF pp. 4–5, Tables I–II.
Magnesium was more sensitive around the optimum tested in their PANOx-SP system. Increasing added magnesium glutamate from 20 to 24 and 28 mM reduced normalized total CAT expression from 1.00 to 0.73 and 0.39. Increasing potassium glutamate from 175 to 200 and 225 mM reduced the corresponding values from 1.00 to 0.94 and 0.92. The magnesium response was therefore sharper in this experiment. Potassium still mattered: at 100 mM potassium glutamate, the relative yield was only 0.63. This supports a formulation-specific sensitivity conclusion rather than a universal statement that potassium is unimportant.
| Solute varied individually | Added concentration, mM | Relative total CAT expression |
|---|---|---|
| Magnesium glutamate | 20 | 1.00 |
| Magnesium glutamate | 24 | 0.73 |
| Magnesium glutamate | 28 | 0.39 |
| Potassium glutamate | 175 | 1.00 |
| Potassium glutamate | 200 | 0.94 |
| Potassium glutamate | 225 | 0.92 |
These are the authors' measurements from Table II, using 15-µL PANOx-SP reactions at 37°C for 3 h, with one solute varied at a time. The magnesium values are additions: approximately 3.3 mM magnesium was also carried over from the extract. Nominal added or total magnesium must not be confused with measured free Mg²⁺. The data concern CAT in the paper's system and do not establish the optimum for sfGFP, TfCut2, or Reteplase. Jewett & Swartz, Table II, PDF p. 4.
Design implication: salt optimization should be considered together with the energy/nucleotide strategy and the defined contributions from the class base buffer, rather than copying a literature value as an additional stock concentration.
The original PANOx-SP formulation adds phosphoenolpyruvate (PEP) as a phosphorylated energy substrate and supplies nucleotide triphosphates: ATP, GTP, UTP, and CTP. These provide nucleotide building blocks and support the energy-demanding transcription/translation system. ATP regeneration is supported by the supplied energy substrate and the enzymes present in the extract. PEP-based reactions can accumulate phosphate and experience pH changes; these effects are part of the chemical environment that must be optimized. Jewett & Swartz, Methods and homeostasis discussion, PDF pp. 2, 5–6.