Team:Jefferson VA SciCOS/Parts

Oxygen promoter-Vitreoscilla hemoglobin (VHb) promoter in E. coli

Vitreoscilla sp. is a gram-negative aerobic bacterium that synthesizes hemoglobin-like molecules (VHb) in response to hypoxic environments, those lacking enough oxygen. This oxygen-dependent VHb molecule is also expressed in E. coli and has shown to improve cellular energetics when in the E. coli strain by using a plasmid-encoded gene as well as single-copy integrants (Khosla 1990). The VHb promoter was used because it is activated under microaerobic conditions, saturated oxygen below a 2% threshold (Khosla and Bailey 1989).

Human basic fibroblast growth factor, bFGF

Human fibroblast growth factor, bFGF- also called FGF-2 and FGF-ß, is a member of fibroblast growth factor family present in extracellular matrixes of blood vessels. During tumor development and wound healing, heparan sulfate enzymes activate bFGF, leading to angiogenesis, the formation of new blood vessels. This recombinant growth factor was use because it has been shown to reduce tissue death and promote improved function after reperfusion, damage to tissue caused by returning bloody supply after an ischemia. bFGF stimulates proliferation of many neuroectodermal, endodermal, and ectodermal cells as well as induces neural differentiation and regeneration. Human bFGF is a 17.2 kDa protein with 154 amino acid residues (Yun, Eun Won, Jeon 2010)

Keratinocyte growth factor, KGF

Keratinocyte growth factor is a growth factor present in the epithelialization phase of wound healing in which an epithelium is formed by keratinocytes, a predominant cell type in the epidermis (Xia, Zhao, Marcus, et al. 1999). This molecule is composed of 194 amino acids and is also known stimulate granulation tissue formation. KGF is able to bind to fibroblast growth factor receptor 2b (FGFR2b) and the complex is dimerized by heparin for signaling to occur (Taylor 2005).

Part created by the TJHSST iGEM team

The goal of the TJHSST iGEM team was to create a part in which the Vitreoscilla hemoglobin promoter is upstream of either the KGF or the FGF producing gene. The promoter is most activated at a threshold of 2% saturated oxygen, which allows for maximum transcription of the downstream gene and production of protein. It is also robustly activated at oxygen thresholds characteristic of hypoxia, but is minimally activated during conditions of anoxia. Essentially, this part is posited to increase production of growth factors at the site of non-healing skin ulcers, such as diabetic gangrene, where the saturated oxygen level borders dangerously low, hypoxic levels. The production of critical growth factors such as FGF at ulcer sites can result in substantial improvement in a short period of time. In a longitudinal study conducted by Asai et al., topical treatment with bFGF and peripheral blood mononuclear cells (PMBC) led to the closing of the ulcer within 6 months. Additionally, the patient was capable of standing and walking by himself after the 6 month time period. As such, topical treatment of a mixture of growth factors and mononuclear cells may be a feasible and effective treatment for hard-to-treat skin ulcers such as diabetic gangrene.

Works Cited

Abraham, J. A., Whang, J. L., Tumolo, A., Mergia, A., Friedman, J., Gospodarowicz, D., Fiddes, J. C. (1986, October). Human basic fibroblast growth factor: nucleotide sequence and genomic organization. The EMBO Jouranl, 5(10), 2523–2528. Retrieved from National Center for Biotechnology Information, NCBI. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1167148/

Asai, J., Takenaka, H., Ichihashi, K., Ueda, E., Katoh, N., Kishimoto, S. (2006, May). Successful treatment of diabetic gangrene with topical application of a mixture of peripheral blood mononuclear cells and basic fibroblast growth factor. National Institutes of Health, 33(5), 349-52. Retrieved from National Center for Biotechnology Information, NCBI. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/16700668

Fox, Sue. WHOLE CELL EXTRACT PREPARATION. Molecular Biology Science Retrieved from http://groups.molbiosci.northwestern.edu/morimoto/research/Protocols/III.%20Proteins/C.%20DNA-Protein%20Interactions/2.%20Whole%20cell%20extract.pdf

Human Keratinocyte Growth Factor(KGF) ELISA Kit. My Bio Source. Retrieved from http://www.mybiosource.com/images/tds/protocol_others/MBS268958.pdf

Khosla, C., Bailey, J. E. (1989, November). Characterization of the oxygen-dependent promoter of the Vitreoscilla hemoglobin gene in Escherichia coli. Journal of Bacteriology, 171(11), 5995–6004. Retrieved from National Center for Biotechnology Information, NCBI. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC210464/

Khosla, Chaitan (1990). Vitreoscilla hemoglobin: gene structure and regulation functions, and applications to aerobic bioprocesses. Dissertation (Ph.D.), California Institute of Technology. Retrieved from http://resolver.caltech.edu/CaltechETD:etd-04212004-154615

Tastan, C. (2009, October). Oxygen promoter-Vitreoscilla hemoglobin(VHb) promoter in E. coli. Igem. Retrieved from http://parts.igem.org/Part:BBa_K258005.

Taylor, Larry P., (2005). FGF 2-FGFR 1c-Heparin (2:2:2) Complex. Retrieved from http://www-personal.umich.edu/~lpt/fgf/1fq9.htm.

Yun, Y., Won, J. E., Jeon, E., Lee, S., Kang, W., Jo, H., Jang, J., Shin, U. S., Kim, H. (2010). Fibroblast Growth Factors: Biology, Function, and Application for Tissue Regeneration. Journal of Tissue Engineering. Retrieved from Sage Journals. Retrieved from http://tej.sagepub.com/content/1/1/218142.full

Xia, Y. P., Zhao, Y., Marcus, J., Jimenez, P. A., Ruben, S. M., Moore, P. A., Khan, F., Mustoe, T. A. (1999, August). National Institutes of Health, 188(4), 431-8. Retrieved from National Center for Biotechnology Information, NCBI. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10440755