Week 2 HW: Final DNA Design

Part 0

  1. Repository name: HA-Neural-Hydrogels
  2. Folder: herrera-ha-neural
  3. Initial construct: Bsub-TuaD-expression-v1

Part 1

What molecule are you expressing? R: TuaD, To investigate whether increasing its expression improves the availability of UDP-glucuronic acid.

What is the sequence? R: I will encode the native tuaD gene from Bacillus subtilis strain 168, which encodes UDP-glucose 6-dehydrogenase TuaD. The reference protein is UniProtKB O32271, also identified as NCBI RefSeq NP_391438.1. The corresponding coding DNA sequence will be retrieved and verified before import into Kernel. Any subsequent codon optimization will preserve the reference amino acid sequence.

Source: UniProtKB O32271.

What host or chassis? The proposed host is an engineered Bacillus subtilis strain. For this TuaD expression module, I assume that the host already expresses a functional hyaluronan synthase (HAS). Increasing TuaD expression is intended to improve the availability of UDP-glucuronic acid, one precursor required for HA synthesis. TuaD alone is insufficient to establish HA production.

Where does the product need to go? TuaD should remain in the cytoplasm, where it converts UDP-glucose into UDP-glucuronic acid. Therefore, I do not propose adding a secretion signal peptide or another targeting sequence. TuaD is the intracellular enzyme; HA is the downstream material intended for recovery from the culture broth.