Difference between revisions of "Part:BBa K1405004"
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<p>We did both agar assay and capillary assay to detect the response of <i>E.coli</i> to different attractants and different concentrations of each attractant. Because the agar assay (Fig.3) is difficult to replicate and collect the data from, we just show the results of capillary assay (Fig.4) . We made a negative control using washing buffer and five concentration gradients (100mM/10mM/1mM/0.01mM/0.0001mM) of attractants. These <i>E.coli</i>s 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).</p>
 
<p>We did both agar assay and capillary assay to detect the response of <i>E.coli</i> to different attractants and different concentrations of each attractant. Because the agar assay (Fig.3) is difficult to replicate and collect the data from, we just show the results of capillary assay (Fig.4) . We made a negative control using washing buffer and five concentration gradients (100mM/10mM/1mM/0.01mM/0.0001mM) of attractants. These <i>E.coli</i>s 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).</p>
 
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<a title="Fig.3  Agar assay" href="https://static.igem.org/mediawiki/2014/7/70/Bnu_cb7.jpg" rel="prettyPhoto"> <span class="overlay zoom" style="display: none;"></span><img style="opacity: 1; width:40%; float: left; margin-left: 50px; border-radius: 0.5em 0.5em 0.5em 0.5em;"src="https://static.igem.org/mediawiki/2014/7/70/Bnu_cb7.jpg"></a>
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<div style="float:left; margin-left: 20px; margin-top: 40px; width: 40%; padding: 30px 20px 30px 30px;"><p class="fig">Fig.3 Agar assay: The three plates shows the chemotaxis results of 8F towards three kinds of attractants, malate, citrate and succinate from left to right. The filter paper of attractants is put on the left, the PBS is put on the right, bacteria is in the middle. Here, 8F swims fastest to malate. </p></div>
 
<div style="float:left; margin-left: 20px; margin-top: 40px; width: 40%; padding: 30px 20px 30px 30px;"><p class="fig">Fig.3 Agar assay: The three plates shows the chemotaxis results of 8F towards three kinds of attractants, malate, citrate and succinate from left to right. The filter paper of attractants is put on the left, the PBS is put on the right, bacteria is in the middle. Here, 8F swims fastest to malate. </p></div>

Revision as of 15:07, 11 October 2014

McfR

Background

McfR is a TCA intermediates responsive chemoreceptor that originally found in Pseudomonas putida. BBa_K1405004 contains McfR coding sequence, an insulator, RBS sequence, and the constitutive promoter BBa_J23100, which is similar to BBa_K515102 that can help us compare our work with iGEM11_Imperial_College _London had done.

This device is used as an additional chemoreceptor for endogenous chemotaxis system in E. coli and responds to L(-) malic acid (HO2CCH2CHOHCO2H) and succinate (HOOC-(CH2)2-COOH).

A 15 bp insulator sequence upstream of the RBS ensures tunability of expression through easy switching of promoters. In addition it allows the translation initiation rate (TIR) of the RBS to remain the same, when the promoter is replaced.

This device is compatible with motile strains of E. coli. It has been transformed and tested in E. coli BL21 in the vector backbone pSB1C3.

Results

We did both agar assay and capillary assay to detect the response of E.coli to different attractants and different concentrations of each attractant. Because the agar assay (Fig.3) is difficult to replicate and collect the data from, we just show the results of capillary assay (Fig.4) . 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).


<img style="opacity: 1; width:10%; border-radius: 0.5em 0.5em 0.5em 0.5em;"src="Bnu_cb7.jpg">

Fig.3 Agar assay: The three plates shows the chemotaxis results of 8F towards three kinds of attractants, malate, citrate and succinate from left to right. The filter paper of attractants is put on the left, the PBS is put on the right, bacteria is in the middle. Here, 8F swims fastest to malate.



<a title="Fig.4 E. coli’s ability of chemotaxis towards different concentrations of succinate or malate." href="Bnu_delivery3.png" rel="prettyPhoto"> <img style="opacity: 1; width:80%; margin-left: 100px;" src="Bnu_delivery3.png"> </a>

Fig.4 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.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1274
  • 1000
    COMPATIBLE WITH RFC[1000]