Part:BBa_K3888011:Design

[Ni-Fe] Hydrogenase Core gene HoxG1 and HoxK1

Figure 1: The Oxygen-tolerant Hydrogenase introduction plasmid constructed, which has a length of 8261bp.

Two parts, HoxG1 and HoxK1, which are the main components of the whole plasmid. HoxG1 encodes the large subunit of the oxygen-tolerant hydrogenase and HoxK1 encodes the small subunit of the oxygen-tolerant hydrogenase.
The position of two parts are located from 5094bp to 6926bp and from 7137bp to 8102bp respectively.
The two parts are promoted by pTac. pTac is controlled by a lac operator with a LacI upstream. IPTG will induce lacI repressors to change its conformation and prevent it from combining with lac operator thus starts the transcription of genes, which are HoxG1 and HoxK1. There is an RBS located between 5068bp and 5087bp. We chose RBS:B0032m as our ribosome binding site. It's a wild-type (also referred to as the natural type).

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 363
  Illegal XbaI site found at 465
  Illegal SpeI site found at 531
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 531
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 678
  Illegal BamHI site found at 1593
  Illegal BamHI site found at 2932
  Illegal XhoI site found at 1915
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 363
  Illegal XbaI site found at 465
  Illegal SpeI site found at 531
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 363
  Illegal XbaI site found at 465
  Illegal SpeI site found at 531
  Illegal AgeI site found at 1809
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

Change signal sequence of HoxK1

Source

Hydrogenovibrio marinus MH-110

References

1. Kim, Jaoon YH, Byung Hoon Jo, and Hyung Joon Cha. "Production of biohydrogen by heterologous expression of oxygen-tolerant Hydrogenovibrio marinus [NiFe]-hydrogenase in Escherichia coli." Journal of biotechnology 155.3 (2011): 312-319.