Part:BBa_K515102

J23100 promoter - PA2652

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 710
  Illegal NgoMIV site found at 812
  Illegal AgeI site found at 118
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1019
  

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 system 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.

Characterisation

Quantitative analysis

A capillary assay was used to quantify the chemotactic response of cells containing the PA2652 construct towards different concentrations of L(-)malic acid. Cells were counted using CFU (colony forming units) and the data obtained were normalised to a number of cells per mL. Capillaries used to expose attractant concentrations were inserted into a bacterial suspension containing 6x10⁸ cells/mL.

Figure 1: Dependence of bacterial chemotaxis to varied malate concetrations. Cells containing PA2652 (BBa_K515102) have shown inreased number of cells in capillaries with 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. The cell count for negative control in each of the capillaries with increasing attractant concentration has not increased. Data collected by Imperial iGEM 2011.

The data shows the ability of cells with the PA2652 construct to perform chemotaxis towards malate. It also depicts varying responses of cells expressing the PA2652 construct to increasing attractant concentrations. From the graph it can be seen that the optimal concentration for rewired chemotaxis towards malate of the E. coli with PA2652 is 1 mM. The sharp decrease in number of cells after 1 mM concentration is caused by malate saturation of the chemoreceptors and the medium. Cells without the construct were unable to perform chemotaxis towards malate and therefore the cell count did not change with increasing attractant concentration.

Behavioral analysis

Behavioral 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 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.

From the data analysis, we can tell 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.