View source for Part:BBa K515102

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<partinfo>BBa_K515102 short</partinfo>


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===Usage and Biology===

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<span class='h3bb'><b>Sequence and Features</b></span>
<partinfo>BBa_K515102 SequenceAndFeatures</partinfo>

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===Functional Parameters===
<partinfo>BBa_K515102 parameters</partinfo>
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<p><b>This BioBrick has been sequence verified.</b>
<h2>Background</h2>
<p>PA2652 is a malate responsive chemoreceptor originally found in <i>Pseudomonas aeruginosa</i> PA01<sup>[1]</sup>.  <a href="https://parts.igem.org/Part:BBa_K515002">BBa_K515002</a> contains an insulator, RBS sequence, and PA2652 coding sequence and its expression is under the control of the constitutive promoter <a href="https://parts.igem.org/Part:BBa_J23100">BBa_J23100</a>. This device is used as an additional chemoreceptor for endogenous chemotaxis in <i>E. coli</i> and responds to L(-)malic acid (HO2CCH2CH(OH)CO2H).</p>
<p>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.</p>
<p>This device is compatible with motile strains of <i>E. coli</i>. It has been transformed and tested in <i>E. coli</i> DH5α in the vector backbone <a href="https://parts.igem.org/Part:pSB1C3">pSB1C3</a>.</p>
<h2>Experimental Data</h2>
<b>Quantitative analysis</b>
<p>Capillary assay was used to quantify the chemotactic response of cells containing PA2652 construct towards different concentrations of L(-)malic acid. Cells were counted using CFU and the data obtained were normalised to a number of cells per mL. Capillaries used to expose attractant concentrations were inserted into the bacterial suspension with 6x10<sup>8</sup> cells/mL.</p>
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<img class="border" src="https://static.igem.org/mediawiki/parts/2/22/ICL_CFU_final_graph.png" width="900px" />
<p><i>Figure 1: Dependence of bacterial chemotaxis to varied malate concetrations. Cells containing PA2652 (BBa_K515102) have shown inreased number of cells in capillaries wiith inreasing malate concentration, with a peak at 1 mM. The number of cells drops sharply after 1 mM due to saturation. Negative control were cells without contruct. These cells have failed to respond to any of the malate concentrations. Data collected by Imperial iGEM 2011.</i></p>
<b>Behavioral analysis</b>
<p>Behavioural analysis of <i>E. coli</i> DH5α was used to characterise the funcionality of this device. This analysis is based on the uniformity of the chemotactic response of a particular population. When bacteria are capable of sensing L(-)malic acid, their behavioural response should be more uniform than that of cells which are unable to sense the chemoattractant. Therefore their velocity is dictated by smooth swimming or random tumbling becomes uniform as the attractant is sensed.</p>
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<img class="border" src="https://static.igem.org/mediawiki/parts/2/26/ICL_PA2652_probability_density_function.png" width="900px" />
<p><i>Figure 2: Probability density function of bacterial number at observed velocities. PA2652 cells exposed to 10 mM malate are more than 90% likely to be moving at just over 2 μm/s. PA2652 cells that were exposed to serine had 90% probability to be moving at a velocity of just over 2 μm/s. PA2652 cells that were not exposed to attractant were had a probability higher than 70%  to be moving at a speed of 2 μm/s. Cells without the BBa_K515102 construct were less than 50% likely to be moving at a velocity of 2 to 4 μm/s. The data depicts a difference in response between PA2652 cells that were and were not exposed to an attractant. In addition, cells without the construct exhibit a lack of uniform response when exposed to 10 mM malate. Data collected by Imperial iGEM 2011.</i></p>
<p>From the data analysis it seems that the bacteria with construct BBa_K515102, when in 10 mM malate, exhibit very uniform behaviour and are therefore recognising the attractant. This is also confirmed by positive control cells exposed to 10 mM serine, where the response of cells is also highly uniform. Cells with the PA2652 construct not exposed to saturating attractant show less uniform movement than PA2652 cells, whether exposed to malate or serine. In addition, negative control cells fail to show uniformity of movement, suggesting inability to recognise the saturating medium with 10 mM malate and performing their movement randomly.</p>
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<h2>References</h2>
<p>[1] Alvarez-Ortega C and Harwood CS (2007) Identification of malate chemoreceptor in <i>Pseudomonas aeruginosa</i> by screening for chemotaxis defects in an energy taxis-deficient mutant. <i>Applied and Environmental Microbiology</i> <b>73</b> 7793-7795.</p>
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