Coding

Part:BBa_K2602000

Designed by: Adham Sakhnini and Julián David Rivera   Group: iGEM18_Lund   (2018-06-05)


Wild type Vitreoscilla haemoglobin (GenBank L21670.1)

Hemoglobin from Vitreoscilla.
Short description: This is part contains the Vitreoscilla hemoglobin wild type coding sequence.


Long Description:
This part contains the coding sequence of Vitreoscilla hemoglobin (VHb) from Vitreoscilla sp. strain C1 (GenBank accession number L21670.1). VHb is a protein found in the aerobic bacterium Vitreoscilla spp. It has been proposed that its main role is to deliver oxygen to terminal respiratory oxidases and thereby increase respiration when the oxygen availability is low, and it may also function as a terminal oxidase itself [1]. The gene coding for VHb (vgb) was first cloned in 1988 [2][3], and has since then been successfully expressed in various organisms such as bacteria [2][3], yeast [4] and even plants [5]. Several studies have shown that expression of vgb can improve cell growth [3][4][6] as well as increase the production of, among other things, enzymes [7][8], antifungals [9] and ethanol [10].

VHb is in its native host a dimer with two protoheme IX molecules [11]. Each of the identical subunits consists of 146 amino acid residues, with a total weight of 15.8 kDa per subunit [12]. VHb has been shown by immunogold electron microscopy to reside in the cytoplasm, to a large extent near the inner cell membrane, in both Vitreoscilla and E. coli [13]. When VHb expressed in E. coli binds a phospholipid, the in vitro oxygen affinity decreases more than 20-fold [14] and it has been suggested that this allows VHb to deliver the oxygen to the respiratory chain [15].

The part was evaluated in the composite parts BBa_K2602010, BBa_K2602011, BBa_K2602013, BBa_K2602014, BBa_K2602015 and BBa_K2602016 expressed in pSB1C3 backbone (part BBa_K1321300). The composites have the same RBS (BBa_B0034), terminators (BBa_B0012 and BBa_B0011) but differs in the promoter (BBa_J23100, BBa_J23101, BBa_J23116, BBa_J23112, BBa_J23113, BBa_K23110); measured strengths from 0 to 1.
The six composite parts were tested to determine the optimal strength of the promoter that increases the production of biomass. The results of the essays show that VHb does not increase the production of biomass during the exponential phase no matter the strength of the promoter. Therefore neither the strength of the promoter nor the Vhb have any effect on the production of biomass during the exponential growth phase in E coli.


Source:
GenBank L21670.1
References:

[1] Stark, B. C., Dikshit, K. L. and Pagilla, K. R. (2012). The Biochemistry of Vitreoscilla hemoglobin. Computational and Structural Biotechnology Journal 3, e201210002. http://doi.org/10.5936/csbj.201210002

[2] Dikshit, K., and Webster, D. (1988) Cloning, characterization and expression of the bacterial globin gene from Vitreoscilla in Escherichia coli. Gene 70, 377-386.

[3] Khosla, C., and Bailey, J. (1988) Heterologous expression of a bacterial haemoglobin improves the growth properties of recombinant Escherichia coli. Nature 331, 633-635.

[4] Wu, JM. and Fu, WC. (2012) Intracellular co-expression of Vitreoscilla hemoglobin enhances cell performance and β-galactosidase production in Pichia pastoris. J Biosci Bioeng 113(3), 332–337.

[5] Holmberg, N., Lilius, G., Bailey, J., and BĂźlow, L. (1997) Transgenic tobacco expressing Vitreoscilla hemoglobin exhibits enhanced growth and altered metabolite production. Nature Biotechnology 15, 244-247.

[6] Pablos, T. E., Mora, E. M., Le Borgne, S., Ramírez, O. T., Gosset, G. and Lara, A. R. (2011), Vitreoscilla hemoglobin expression in engineered Escherichia coli: Improved performance in high cell‐density batch cultivations. Biotechnology Journal, 6: 993-1002. doi:10.1002/biot.201000405

[7] Khosravi, M., Webster D. A. and Stark, B.C. (1990). Presence of the bacterial hemoglobin gene improves Îą-amylase production of a recombinant Escherichia coli strain. Plasmid 24(3), 190-194.

[8] Wu, JM., Wang, SY., Fu, WC. (2012). Lower Temperature Cultures Enlarge the Effects of Vitreoscilla Hemoglobin Expression on Recombinant Pichia pastoris. Int. J. Mol. Sci. 13(10), 13212-13226; doi:10.3390/ijms131013212

[9] Wang S, Liu F, Hou Z, Zong G, Zhu X, Ling P. Enhancement of natamycin production on Streptomyces gilvosporeus by chromosomal integration of the Vitreoscilla hemoglobin gene (vgb) World J Microb Biot. 2014;30:1369–1376. doi: 10.1007/s11274-013-1561-4

[10] Sanny, T., Arnaldos, M., Kunkel, S.A. et al (2010). Engineering of ethanolic E. coli with the Vitreoscilla hemoglobin gene enhances ethanol production from both glucose and xylose. Appl Microbiol Biotechnol 88, 1103-1112. https://doi.org/10.1007/s00253-010-2817-7

[11] Webster, D. A. and Hackett, D. P., 1966. The Purification and Properties of Cytochrome o from Vitreoscilla. The Journal of Biological Chemistry, 241(14) pp. 3308-3315

[12] Wakabayashi S, Matsubara H and Webster DA. (1986). Primary sequence of a dimeric bacterial haemoglobin from Vitreoscilla. Nature, 322 pp. 481-483

[13] Ramandeep, Wang KW, Raje M, Kim K, Stark BC, Dikshit KL, Webster DA. (2001). Vitreoscilla Hemoglobin intracellular localization and binding to membranes. Journal of biological chemistry, DOI 10.1074/jbc.M009808200.

[14] Rinaldi AC, Bonamore A, Macone A, Boffi A, Bozzi A, Di Giulio A. (2006). Interaction of Vitreoscilla Hemoglobin with Membrane Lipids. Biochemistry, 45, 4069-4076.

[15] Anand A, Duk BT, Singh S, Akbas MY, Webster DA, Stark BC, Dikshit, KL. (2010). Redox-mediated interactions of VHb (Vitreoscilla haemoglobin) with OxyR: novel regulation of VHb biosynthesis under oxidative stress. Biochemical Journal, 426(3) 271-280.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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