Results

E. coli Environment Alters Tumbling:

Bacteria are sensitive to a number of factors within the growth medium. Salt is a particularly important component of the medium, but concentrations above or below optimal levels can have a dramatic impact on growth and survival. The power of our engineered system is that sub-optimal environmental conditions can be measured in real time. We tested our system in the presence of varying concentrations of NaCl and measured the tumbling response after overnight growth.

Salt Experiment

CONTROL

0.0 M NaCl

RPS (rotations/sec) 0.21 0.17 0.48 0.71 0.45
Frequency (Hz) 132 107 302 446 283

0.5 M NaCl

RPS (rotations/sec) 0.80 0.83 0.72 0.71 0.71
Frequency (Hz) 503 522 452 446 446

0.9 M NaCl

RPS (rotations/sec) 0.21 0.55 0.71 0.40 0.54
Frequency (Hz) 132 346 446 251 339

CheY

0.0 M NaCl

RPS (rotations/sec) 0.57 0.60 0.68 0.78 0.74
Frequency (Hz) 358 377 427 490 465

0.5 M NaCl

RPS (rotations/sec) 0.32 0.40 0.43 0.25 0.35
Frequency (Hz) 201 251 270 157 220

0.9 M NaCl

RPS (rotations/sec) 0.31 0.32 0.21 0.25 0.28
Frequency (Hz) 195 201 132 157 176

CheZ

0.9 M NaCl

RPS (rotations/sec) 0.63 0.50 0.25 0.63 0.31
Frequency (Hz) 396 314 157 395 195

CheY vs. CONTROL

Salt Concentration 0.0M NaCl 0.5M NaCl 0.9M Nacl
Control CheY Control CheY Control CheY
Avgerage frequency (Hz)

254

424

474

220

303

172
Standard Deviation

138

56

36

44

118

28
Two-tailed t-test (a=0.05)

0.0127

0.0003

0.0978

CheY vs. CheZ vs. CONTROL

Control cheY cheZ
Avgerage frequency (Hz) 303 172 292
Standard Deviation 28 118 115
Two-tailed t-test (a=0.05) 0.0978 0.9136

Inducible Tumbling System in cheY and cheZ

In order to better control the tumbling/swimming response in E. coli, we generated biobricks that facilitate the overexpression of cheY and cheZ, which directly influence clockwise/counterclockwise movement of the flagella. To test our bacterial communication system, we transformed cheY and cheZ parts into E. coli B strain HT115. Our BioBricks included an inducible T7 promoter that is activated by IPTG. As shown in the figure, 100% of successfully transformed bacteria with the T7 RBS cheY construct showed tumbling, demonstrating the functionality of the T7 RBS cheY part.

Reconstitution of Flagella

Our team created the following BioBricks that control the assembly and operon regulation of E. coli flagella: BBa_K842000 flhB, BBa_K842001 flhD, BBa_K842002 flgJ, BBa_842003 fliA, BBa_K842005 fliH, BBa_K842011 motA, BBa_K842012 motB. These parts allow for the reconstitution of a flagella mechanism in E. coli B strains where the flagella mechanisms are lacking.



Future Directions

We have begun also characterizing the tumbling response of E. coli to acidic and basic growth conditions. These data are preliminary as we have only tested a small range of pH and have yet to test our inducible cheY and cheZ systems.

pH Experiment

CONTROL

pH 5

RPS (rotations/sec) 0.25 0.33 0.36 0.20 0.42
Frequency (Hz) 157 207 226 126 264

pH 6

RPS (rotations/sec) 0.43 0.36 0.46 0.40 0.25
Frequency (Hz) 270 226 289 251 157

pH 7

RPS (rotations/sec) 0.21 0.17 0.48 0.71 0.45
Frequency (Hz) 132 107 302 446 283

pH 8

RPS (rotations/sec) 0.25 0.50 0.25 0.35 0.33
Frequency (Hz) 157 314 157 220 207

pH 9

RPS (rotations/sec) 0.26 0.25 0.33 0.33 0.32
Frequency (Hz) 163 157 207 207 201

pH 5 6 7 8 9
Average frequency (Hz) 196 238 254 211 187
Standard Deviation 55 51 138 64 24