Part:BBa_K515102:Experience
User Reviews
UNIQfd321ac07cb756ac-partinfo-00000000-QINU
No review score entered. iGEM14_BNU-China |
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. ResultsWe 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. |
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UNIQfd321ac07cb756ac-partinfo-00000004-QINU
No review score entered. iGEM14_BNU-China |
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. ResultsWe 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. |
UNIQfd321ac07cb756ac-partinfo-00000007-QINU