Difference between revisions of "Part:BBa K515102"

Revision as of 03:54, 22 September 2011

J23100 promoter - PA2652

Sequence and Features

This BioBrick has been sequence verified.

Background

PA2652 is a malate responsive chemoreceptor originally found in Pseudomonas aeruginosa PA01^[1]. BBa_K515002 contains an insulator, RBS sequence, and PA2652 coding sequence and its expression is under the control of the constitutive promoter BBa_J23100. This device is used as an additional chemoreceptor for endogenous chemotaxis in E. coli and responds to L(-)malic acid (HO2CCH2CH(OH)CO2H).

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 DH5α in the vector backbone pSB1C3.

Experimental Data

Behavioural analysis of E. coli 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.

Figure 1: 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.

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.

References

[1] Alvarez-Ortega C and Harwood CS (2007) Identification of malate chemoreceptor in Pseudomonas aeruginosa by screening for chemotaxis defects in an energy taxis-deficient mutant. Applied and Environmental Microbiology 73 7793-7795.