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

Malate responding chemoreceptor originally found in Pseudomonas aeruginosa PA01^[1] is a codon optimised translational subunit. This subunit BBa_K515002 contains optimised insulator and RBS sequence, its expression is under the control of constitutive promoter BBa_J23100. Device is used as an additional chemoreceptor for endogenous chemotaxis mechanism of E. coli. Device responds to L (-) malic acid, linear formula (HO2CCH2CH(OH)CO2H).

Device contains 15 bp insulator seuence, which ensures tunability of expression through easy switching of promoters. In addition, insulator sequence allows the translation initiation rate (TIR) of the ribosome binding site (RBS) to remain the same, when the promoter is replaced.

Device is compatible for motile strains of E. coli. It has been tested in E. coli DH5α strain, inserted in the vector backbone pSB1C3.

Experimental Data

Behavioral analysis of E. coli cells was used to identify functioning of this device. The analysis is based on the transient difference in velocities of bacteria capable of sensing chemoattractant, when exposed to attractant which is in comparison to control cells, that are unable to sense L (-) malic acid.

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 were 90% likely to be moving at velocity just over 2 μm/s. PA2652 cells that were not exposed to attractant were over 70% likely to be moving at 2 μm/s. Cells without BBa_K515102 construct were less than 50% likely to be moving at velocity between 2 and 4 μm/s. Data depicts difference in response between PA2652 cells, which were and which were not exposed to an attractant. Also cells without construct show lack of uniform response when exposed to 10 mM malate. Data collected by Imperial iGEM 2011.

Bacteria with construct BBa_K515102, when in 10 mM malate follow the trend of positive control cells, which were exposed to 10 mM serine. The cells containing construct with probable to be in a

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.