Difference between revisions of "Part:BBa K3656307"
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Revision as of 18:40, 27 October 2020


OT3-Carbonic Anhydrase-Mutant-His

This is an improved part of mutant carbonic anhydrase from Pyrococcus horikoshii OT3 which converts the incoming bicarbonate into carbon dioxide in the carboxysome, a step that is essential for CO2 fixation. We have optimized codon sequence in the coding sequence (CDS) to make the mutant carbonic anhydrase gene more suitable for expression in E. coli. In addition to codon optimization, we have added a His-tag to the C-terminal of the CDS that is used for purification of carbonic anhydrase protein.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 470
    Illegal XhoI site found at 520
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Construction of OT3-CA2-MU-His expression plasmid

By using the software Autodock for molecular docking, we studied the docking conformation of the substrate at the catalytic site, and analyzed the interaction between the residue at the catalytic site and the substrate. Autodock and PyMOL were used to further investigate the effects of secondary and tertiary structures of catalytic sites on catalytic processes. After research and discussion, we set up the mutation site and the alternative residue scheme, carried on the molecular docking of the recombinant enzyme, and then compared the enzyme-substrate docking conformation before and after the reorganization. Finally, suitable mutation sites and alternative residues were selected to simulate the mutation and the catalytic performance was improved theoretically.

Based on the simulation results, we finally determine appropriate mutations are respectively: 122: P→K (the 122nd proline is mutated to lysine), position 123: P→K (the 123rd proline is mutated to Lysine), position 128: P→K (mutation of the 128th proline to lysine), position 132: L→K (mutation of 132nd leucine to lysine), position 134: L→K ( Leucine at position 134 was mutated to lysine), and position 137: P→K (leucine at position 137 was mutated to lysine), and finally a mutant OT3-CA (OT3-CA-MU, BBa_K3656307, Fig.9 and 10) to enhance its catalytic activity under high-temperature conditions was obtained (Fig. 1).

Fig. 1 Structure of Mutant OT3-CA
Table 1. Using Auto Dock software to analyze the docking results of OT3-CA-WT and OT3-CA-MU with carbonic acid respectively.
Name OT3-CA-WT OT3-CA-MU
Part Number BBa_K3656305 BBa_K3656309
binding_energy -5.08 -5.37
ligand_efficiency -1.27 -1.34
inhib_constant 187.83 116.58
inhib_constant_units uM uM
intermol_energy -5.68 -5.96
vdw_hb_desolv_energy -1.76 -1.92
electrostatic_energy -3.92 -4.04
total_intermal 0.05 0.02
torsional_energy 0.6 0.6
unbound_energy 0.05 0.02

Secondly, we also use SnapGene design software to simulate and construct OT3-CA-MU-His recombinant vector (Fig. 2 with pET-28a (+) as the carrier.

Fig. 2 Map of OT3-CA-MU-His recombinant vector