Part:BBa_K117008:Experience

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Applications of BBa_K117008

This pLsrA-YFP part aims to characterize the LsrA promoter (Part K117002).

User Reviews

UNIQ3a23c29f1d3d5551-partinfo-00000000-QINU UNIQ3a23c29f1d3d5551-partinfo-00000001-QINU Team Michigan 2010: We attempted to characterize this part in E. coli DH5α and in a LuxS^- strain of E. coli. We failed to replicate the Team Singapore-NTU's results; however, our positive control reporter strains also failed to show induction of GFP activity in response to AI-2, so we believe that there is some problem with our experimental protocols. See Michigan 2010 wiki for more details. Team:Michigan

Microplate Reader Data:

Preparation of AI-2 for characterization:

Pure AI-2 is very difficult to harvest or synthesize. However, as a quorum sensing messenger, AI-2 is present in intercellular medium in considerable amount. In this project, AI-2 was indirectly harvested by obtaining cell supernatants at different time points of cell growth. Glucose was added to the growth medium as it promotes AI-2 production significantly (Liang Wang, 2005).

A colony of E. coli W3110 was grown overnight in LB for 16 hours at 37°C . The overnight culture was then diluted in LB plus 0.8% glucose to an optical density at 600 nm (OD600) of 0.02. The cells were incubated at 37°C with shaking at 225 rpm in 500ml Erlenmeyer flask. Cell aliquots were removed at each time point for measurement of the OD600. The measured values were shown in the following table:

Time (hrs)	OD of W3110
0	0.002
1	0.047
2	0.112
3	0.341
4	0.628
5	0.921
6	1.032
7	1.172
8	1.252

Based on the data, a graph of OD600 versus time was constructed as below:

OD of W3110 vs time

Maximal AI-2 production activity is typically observed during mid- to late exponential phase (Surette, 1998). Therefore, 4ml of samples corresponding to 2, 3, 3.333, 3.667, 4, 6 time points were collected and centrifuged at 13,200 rpm for 10 min in a microcentrifuge to separate cell pellets from supernatants. Cleared supernatants were filtered using 0.2 µm pore size filters to remove unwanted big proteins. Supernatants were stored at -20°C.

Bacteria strains and media

Bacteria used in all the experiments were LuxS mutants derived from the wild type E coli strain W3110. By knocking out LuxS gene, the cells were unable to synthesize AI-2. As a result, lsrA promoter could only be induced in the presence of external AI-2 produced by other bacteria rather than the host cells. LuxS mutants were first made chemically competent in order to be able to take in biobrick plasmids during transformation. The media used for overnight growth of cells were Luria-Bertani broth (LB). Cells were diluted in M9 media to an OD600 of 1 before ready to be read under Fluorescence Microplate Reader or Absorbance reader.

pLsrA-lysis plasmid (part BBa_K117010) in normal W3310 E.coli.(left) and LuxS mutants (right): In the first case, the bacteria produce their own AI-2 to cause themselves to lyze

Characterization:

Graph of yellow fluorescence unit versus time: For the control samples with only cells, the RFU remained at a steady level with time. The same thing occurred for the cells with 50µl LB broth added, although RFU level was a bit higher. For the cells with 50µl supernatants containing AI-2, an increasing trend in the RFU can be clearly observed.

References:

• Liang Wang, Yoshifumi Hashimoto, Chen-Yu Tsao, James J.Valdes, and William E.Bentley,Cyclic AMP (cAMP) and cAMP receptor protein influence both synthesis and uptake of extracellular Autoinducer 2 in Escherichia coli,Journal of Bacteriology,2005,2066-2076.
• Surette, M. G., and B. L. Bassler,Quorum sensing in Escherichia coli and Salmonella typhimurium,Proc. Natl. Acad. Sci.,1998,7046-7050.