Part:BBa_K1321200

J23101+B0034+VHb (Vitreoscilla haemoglobin)

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.

Expressing BBa_K1321200 in pSEVA331-Bb backbone (part BBa_K1321300) in the cellulose-producing Gluconacetobacter 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 Figure 1).

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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. **** denotes 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.

Updated Sep. 2016

To measure the characters of Vitreoscilla haemoglobin generator constructed by iGEM14_Imperial (BBa_K1321200). Vitreoscilla haemoglobin can promote the cell growth and protein production ability. We ligate mRFP generator (BBa_J04450) with VHb generator together (BBa_K1919500) to test the protein production ability in E. coli.

In iGEM14_Imperial project, the VHb is used in Gluconacetobacter xylinus strain to promote the production of cellulose. SCU-China 2016 are expected to express this gene in E. coli to promote the engineered bacteria growth ability in relatively bad condition (Shoes, for example). Except observational measurement, we did the quantitative measurement in different oxygen concentrations to test its ability.

In observational measurement, we use two E. coli BL21 strains which respectively contains mRFP (BBa_J04450) and mRFP + VHb (K1919500) to compare their growth and mRFP production by naked eyes. The result obviously shows that the colony is bigger and contains more mRFP.

Figure 1. Left is E. coli BL21 strain only contains mRFP. Right is E. coli BL21 strain contains mRFP plus VHb. The colony right is bigger and contains more mRFP.

In quantitative measurement, we use different oxygen concentrations (created by gas production pack) to test if VHb can really work. Because the synthesis of VHb cost energy. But if the volume of energy cost is lower than what VHb can bring, as a result, VHb makes the strain grow slower. We think oxygen concentration is an important factor. And we raise a theory called “VHb energy cost theory” in our modeling part. The result is not shown in the part information page, please visit our wiki (SCU-China 2016).

Figure 2a. OD600 test shows in 20ml/50ml in different oxygen growth condition, the tendency is similar. It may be due to the high volume liquid culture in tubes and E. coli process anaerobic respiration.

Figure 2b. Fluorescence result show the same tendency as Figure 2a.

Figure 2b. Figure 2c. OD600/fluorescence result show the same tendency as Figure 2a.

Figure 3a. OD600 continuous test in 96 wells plate in air shows the similar result to 20ml/50ml test. It may be still due to the anaerobic respiration.

'Figure 3b. Fluorescence result shows the same tendency as Figure 3a.

’Figure 3c. OD600/fluorescence results show the opposite tendency to Figure 3a.'

Figure 4a. 5ml/50ml and 20ml/50ml cultivated in air OD600 test shows VHb does accelerate the growth of E. coli in early time for small volume cultivation. But at the end, because of the energy cost on VHb, strain contains mRFP + VHb has lower carrying capacity K.

Figure 4b. Fluorescence result shows the same tendency as Figure 4a.

Figure 4c. OD600/fluorescence results show the opposite tendency to Figure 4a.

Figure 5a. 5ml/50ml cultivated in different oxygen concentrations at 6h in OD600 test shows VHb does accelerate the growth of E. coli in the existence of oxygen. '

Figure 5b. Fluorescence result shows the opposite tendency as Figure 5a.

In conclusion, VHb can accelerate the growth and improve protein production in some condition. For more details and analysis, please visit wiki of SCU-China 2016.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 477
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]