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Part:BBa_K515102:Experience

Designed by: Atipat Patharagulpong   Group: iGEM11_Imperial_College_London   (2011-09-07)
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The plants root exudates contain TCA intermediates that can attract bacteria having the ability of chemotaxis. E.coli has five kinds of chemoreceptors, which interact with factors of the flagella that leads to chemotaxis. But E.coli doesn’t have specific chemotaxis towards some TCA intermediates while Pseudomonas putida has some McpS, like McfQ and McfR. We made a part BBa_K1405004 containing the sequence of McfR, which detects succinate, malate and fumarate. Then we detected its chemotaxis towards malate and succinate, and did the same assay to BBa_K515102. What's more, we changed the chassis to BL21, which expresses better than DH5α they had used.


Results

We did capillary assay to detect the response of E.coli to different attractants and different concentrations of each attractant. We show the results of capillary assay (Fig.1) below. We made a negative control using washing buffer and five concentration gradients (100mM/10mM/1mM/0.01mM/0.0001mM) of attractants. These E.colis were divided into three groups based on the plasmid they have been transformed into. The plasmids are biobricks, BBa_K608003 and BBa_K515102 (they are from 5A and 8F wells in plate1), and the McfR plasmid was designed by us. BBa_K608003 (5A) only has a strong promoter and medium RBS, so it doesn’t have specific chemotaxis towards TCA intermediates. BBa_K515102 (8F) is a biobrick from 2011_Imperial_College_London, which responds to L(-)malic acid (HO2CCH2CH(OH)CO2H).







Fig.2 E. coli’s ability of chemotaxis towards different concentrations of succinate or malate.
The cells were diluted 20000 times. 5A is a control which doesn’t have chemotaxis towards malate or succinate.
Malate: McfR showed the strongest respond. The tendency of curve of 8F was biphasic with maximums at attractant concentration of about 10-2 M and 10-5 M, while the tendency of curve of McfR was biphasic with maximums at attractant concentration of about10-2 M and 10 -7 M. Both of them reached minimum at attractant concentration of about 10 -1 M, the number of cells decreased slowly with attractant concentration decreasing for 8F and McfR. There was a significant difference among test and control (p < 0.05), and quantity of cells of 8F and McfR was much more than that of 5A. So both of 8F and McfR have chemotaxis towards malate. And McfR shows stronger response.
Succinate: The tendencies of curves of 8F and McfR towards succinate are same. As the attractant concentration increased, the number of cells arose and reached the maximum at attractant concentration of about 10-2 M and fell sharply with the minimum at attractant concentration of 10-1 M. The quantities of cells of 8F and McfR were almost equivalent and did not have significant difference. But there were significant differences among 8F & 5A, and McfR & 5A: the number of cells of 5A are far less than 8F or McfR (p < 0.05), which demonstrated that 8F and McfR have chemotaxis towards succinate and the capacity of chemotaxis of 8F and McfR towards succinate are almost equal. When compared with malate, 8F shows stronger response, while McfR shows weaker response.



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