Part:BBa_K1321202

VHb in pSEVA321-Bb

VHb (BBa_K1321200) in pSEVA321-Bb (BBa_K1321301)

This part is a hemoglobin isolated from Vitreoscilla (VHb) expressed behind a strong Anderson promoter and a strong RBS. VHb is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[1][2][3][4]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[5], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce, resulting in increased cell metabolism. This part contains a constitute promoter and RBS ready for expression.

The effects of expression of Vhb in pSEVA321-Bb (BBa_K1231301) in the cellulose producing, strickly aerobic bactgerium G.xylinus are undergoing characterization. However expressing Vhb (BBa_K1321200) in pSEVA331-Bb backbone (part BBa_K1321300) in G.xylinus strain igem (part BBa_K1321306; grown at 30degC 180rpm in 5ml HS-cellulase medium, in 50ml tubes for 4 days) increases biomass production almost two-fold (see BBa_K1321201).

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Figure 1. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. G.xylinus igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase medium without inoculations) showed no growth (not shown here). N=3 (VHb), 4 (wild-type), error bars denote SD, **** - p=<0.0001.

References:

[1] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli.

[2] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin.

[3] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor.

[4] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin.

[5] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 4084
  Illegal suffix found in sequence at 513
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4084
  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal SpeI site found at 514
  Illegal PstI site found at 528
  Illegal NotI site found at 521
  Illegal NotI site found at 4090
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4084
  Illegal BglII site found at 477
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 4084
  Illegal suffix found in sequence at 514
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 4084
  Illegal XbaI site found at 4099
  Illegal SpeI site found at 514
  Illegal PstI site found at 528
  Illegal NgoMIV site found at 1703
  Illegal NgoMIV site found at 2588
  Illegal NgoMIV site found at 3699
  Illegal NgoMIV site found at 3823
  
• 1000
  COMPATIBLE WITH RFC[1000]