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Oxygen Promoter
From 2009.igem.org
The gene coding for the Vitreoscilla hemoglobin (VHb) molecule has been cloned and functionally expressed in Escherichia coli. By using a plasmid-encoded gene as well as single-copy integrants, the oxygen-dependent VHb gene (VHb) promoter was shown to be functional in E. coli. The promoter was maximally induced under microaerobic conditions (dissolved oxygen levels of less than 2% air saturation).
We wanted to measure the activity of VHb gene; therefore, the VHb promoter. The VHb promoter becomes active when the surrounding oxygen amount lowers to 2%. We wanted to see whether this promoter will effectively work with our E. Coli cells.
Measuring the growth of our E. Coli cells with respect to oxygen availability and VHb gene presence is the main purpose.
In our experiment, we used 4 different groups of bacteria. The first group of bacteria have VHb gene and aerated environment. Second group has only VHb gene and kept in air closed system. The third group doesn’t have VHb gene; Instead they have plasmids only provide resistance to amphicilin. This group is placed in aerated environment. The last group is kept in air closed environment and VHb gene is not transformed. Instead, like the third group, they have transformed only amphicilin resistance plasmid.
As media, Luria-Bertani (LB) broth with amphicilin has been used. In 2 ml eppendorfs, 1.51 ml LB has been added. 0.09 ml overnight culture of E.Coli MK-57 strain with shaking at 320 rpm has been used. 5 samples of each group have been labeled and the unaerated groups have been closed with parafilm. The measurements have been taken in every 1.5 hour. 5 measurements for each has been performed.
Our results can be observed in the graph. The results were appeared as expected. The presence of VHb gene in our E.coli cells have limited the growth because the disappearance of oxygen have started the VHb gene expression and with passing time, the expression of VHb increased. This caused limited growth of our E.coli cells. The smallest growth is seen in E. Coli cells having VHb gene and the unaerated environment.The significance of the difference between the data of groups have been compared with ANOVA and it appeared to be significant.
References
1. Khosla, C. & Bailey, J.E (1989) Characterization of the Oxygen-Dependent Promoter of the Vitreoscilla Hemoglobin Gene in Escherichia coli. Journal of Bacteriology volume 171 pp.5995-6004
2. Geckil H*, Stark BC, and Webster DA(2001)Cell growth and oxygen uptake of Escherichia coli and Pseudomonas aeruginosa are differently affected by the genetically engineered Vitreoscilla hemoglobin gene.Journal of Biotechnology 2001; 85(1): 57-66.
