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

We measured a large variability in the frequency distribution for control growth conditions. We interpret this to mean the E. coli are randomly tumbling and swimming at all times. Intriguingly, under environmental distress the frequency distribution unifies, resulting in a single harmonic. See below.

0.5 M NaCl

0.9 M NaCl

CheY

0.0 M NaCl

Overexpression of cheY produces a frequency equivalent to G#6.

0.5 M NaCl

Overexpression of cheY in the presence of 0.5M NaCl yields a harmonic frequency of A3.

0.9 M NaCl

Overexpression of cheY in the presence of 0.9M NaCl yields a harmonic frequency similar to F3.

We measured a large variability in the frequency distribution for control growth conditions. We interpret this to mean the E. coli are randomly tumbling and swimming at all times. Intriguingly, under environmental distress the frequency distribution unifies, resulting in a single harmonic. See below.

CheZ

0.9 M NaCl

The frequency response in bacteria that overexpress cheZ do not express a statistically significant trend.

CheY vs. CONTROL

CheY vs. CheZ vs. CONTROL

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

pH 6

pH 7

pH 8

pH 9