Difference between revisions of "Part:BBa K515102:Experience"

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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- 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 DH5�α 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.1 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: 8F 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, and reached minimum at attractant concentration of about 10^-1 M, which is similar to others’ work. With attractant concentration decreasing for 8F, the number of cells decreased slowly. McfR stayed stable at range of 10^-7 M to 10^-3 M, and had only one phase which reached its maximum at attractant concentration of 10^-2 M, then it went down sharply at 10^-1 M. There was a significant difference among 8F and 5A (p < 0.05), and quantity of cells of 8F was much more than that of 5A. So 8F has chemotaxis towards malate. On the other hand, McfR and 5A were not significantly different and quantity of cells of McfR was little less than it of 5A. So McfR shows no chemotaxis towards malate here.
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. Compared with chemotaxis towards malate, both of 8F and McfR show stronger chemotaxis towards succinate.