Difference between revisions of "Part:BBa K1321200"

Revision as of 13:32, 2 November 2014

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.

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]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1321200 short</partinfo>
-This part is a haemoglobin 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.
+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).
@@ Line 8: / Line 8: @@
-[[File:IC14 Vhb effects on growth correct.jpg|700px|thumb|center|'''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. ''' ]]
+[[File:IC14 Vhb effects on growth correct.jpg|700px|thumb|center|'''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. ''' ]]