Part:BBa_K1412010
Drive the E.coli engineered strain(CL-1) towards death region
What it is
Composite part enables the engineered bacteria tends to killing osmotic pressure which will kill bacteria itself.
How it works
E.coli make use of the EnvZ/OmpR system to mediate signal transduction in response to environmental osmolarity changes. EnvZ, a histidine kinase, undergoes trans-autophosphorylation, then the high energy phosphoryl group is subsequently transferred to OmpR, a response regulator. In the system, OmpR-controlled promoter (pOmpC) is involved in. The expression strength of pOmpC is depending upon the medium osmolarity. When medium osmolarity is increasing, the EnvZ will phosphorylate more OmpR into phosphorylated OmpR (OmpR-P), and more OmpR-P will result in stronger expression strength of pOmpC. In our circuitry design, CheZ is upstream regulated by pOmpC.
We use semi-solid medium culture with gradient concentration of sucrose to characterize the device (BBa_K1412010). Setting the motile ability is proportional to the moving radius. From the plot, when no sucrose added in, the motile ability is the weakest. The motile ability keeps growing while the concentration of sucrose increased from 0 to 4%. Then the motile ability went down slightly as the sucrose concentration increased from 4% to 10%, but is still stronger than that at concentration 0. We can make the conclusion that our device is working as expectation, the motile ability went down because of the inhibition from hyperosmotic pressure. Besides, for even at the inhibiting osmotic pressure, the motile ability is still stronger than that without any inducer, reprogrammed CL-1 may even tends to killing osmotic pressure which will kill bacteria itself.
Experimental data
Figure 1. The plot of Moving radius versus Sucrose concentration. The four curves were measured after 10 h, 11 h, 12 h and 16.5 h respectively.
Figure 2. Drawing horizontal line with 10% sucrose and erect line with water. Spotting cells on the cross. Two plates are parallel experiment. Culturing for 48 hours, we observed that reprogrammed E.coli has significant orientation to high concentration line. As high concentration sucrose generates high hyperosmosis, it has proved that CL-1 has the tendency swimming to high osmotic pressure.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
[1] [http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2006.05048.x/abstract;jsessionid=DB389E887A1738CF004BAD1B61A6D336.f01t01 Batchelor, Eric, and Mark Goulian. "Imaging OmpR localization in Escherichia coli." Molecular Microbiology 59.6(2006):1767–1778.]
[2] [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC216723/ Kawaji, H., T. Mizuno, and S. Mizushima. "Influence of molecular size and osmolarity of sugars and dextrans on the synthesis of outer membrane proteins O-8 and O-9 of Escherichia coli K-12." Journal of bacteriology 140.3 (1979): 843-847.]
More information, click here: [http://2014.igem.org/Team:XMU-China/Project_Application_BlackHole# XMU-China Black Hole]
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