Team:USC/Project2

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Latest revision as of 03:53, 4 October 2012

Results « E. musici

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

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

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

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

0.5 M NaCl

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

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

0.9 M NaCl

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

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

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

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

CheY vs. CONTROL

Salt Concentration	0.0M NaCl		0.5M NaCl		0.9M Nacl
	Control	CheY	Control	CheY	Control	CheY
Average 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

@@ Line 339: / Line 339: @@
 </tbody>
 </table>
+<p><span style="color:#000000;">We measured a large variability in the frequency distribution for control growth conditions. We interpret this to mean the <em>E. coli</em> are randomly tumbling and swimming at all times. Intriguingly, under environmental distress the frequency distribution unifies, resulting in a single harmonic. See below.</span></p>
+<p><span style="color:#000000;"><strong><span style="text-decoration:underline;"><a href="http://uscigem2012.files.wordpress.com/2012/07/control_00m.png"><span style="text-decoration:underline;color:#000000;"><img class="aligncenter size-full wp-image-287" title="Screen Shot 2012-10-03 at 7.54.06 PM" src="http://uscigem2012.files.wordpress.com/2012/07/control_00m.png" alt=""   /></span></a></span></strong></span></p>
 <p><span style="color:#000000;"><strong>0.5 M NaCl </strong></span></p>
 <table border="1" cellspacing="0" cellpadding="0">
@@ Line 404: / Line 407: @@
 </tbody>
 </table>
+<p><span style="color:#000000;">Overexpression of <em>cheY</em> produces a frequency equivalent to G#6.</span></p>
+<p><span style="color:#000000;"><strong><span style="text-decoration:underline;"><a href="http://uscigem2012.files.wordpress.com/2012/07/chey_00m.png"><span style="text-decoration:underline;color:#000000;"><img class="aligncenter size-full wp-image-287" title="Screen Shot 2012-10-03 at 7.54.06 PM" src="http://uscigem2012.files.wordpress.com/2012/07/chey_00m.png" alt=""   /></span></a></span></strong></span></p>
 <p><span style="color:#000000;"><strong>0.5 M NaCl </strong></span></p>
 <table border="1" cellspacing="0" cellpadding="0">
@@ Line 425: / Line 432: @@
 </tbody>
 </table>
+<p><span style="color:#000000;">Overexpression of <em>cheY</em> in the presence of 0.5M NaCl yields a harmonic frequency of A3.</span></p>
+<p><span style="color:#000000;"><strong><span style="text-decoration:underline;"><a href="http://uscigem2012.files.wordpress.com/2012/07/chey_05m.png"><span style="text-decoration:underline;color:#000000;"><img class="aligncenter size-full wp-image-287" title="Screen Shot 2012-10-03 at 7.54.06 PM" src="http://uscigem2012.files.wordpress.com/2012/07/chey_05m.png" alt=""   /></span></a></span></strong></span></p>
 <p><span style="color:#000000;"><strong>0.9 M NaCl </strong></span></p>
 <table border="1" cellspacing="0" cellpadding="0">
@@ Line 446: / Line 458: @@
 </tbody>
 </table>
+<p><span style="color:#000000;">Overexpression of <em>cheY</em> in the presence of 0.9M NaCl yields a harmonic frequency similar to F3.</span></p>
+<p><span style="color:#000000;">We measured a large variability in the frequency distribution for control growth conditions. We interpret this to mean the <em>E. coli</em> are randomly tumbling and swimming at all times. Intriguingly, under environmental distress the frequency distribution unifies, resulting in a single harmonic. See below.</span></p>
+<p><span style="color:#000000;"><strong><span style="text-decoration:underline;"><a href="http://uscigem2012.files.wordpress.com/2012/07/chey_09m.png"><span style="text-decoration:underline;color:#000000;"><img class="aligncenter size-full wp-image-287" title="Screen Shot 2012-10-03 at 7.54.06 PM" src="http://uscigem2012.files.wordpress.com/2012/07/chey_09m.png" alt=""   /></span></a></span></strong></span></p>
 <p><span style="color:#000000;"><strong><span style="text-decoration:underline;"> </span></strong></span></p>
 <p><span style="color:#000000;"><strong><span style="text-decoration:underline;">CheZ</span></strong></span></p>
@@ Line 469: / Line 488: @@
 </tbody>
 </table>
+<p><span style="color:#000000;">The frequency response in bacteria that overexpress <em>cheZ</em> do not express a statistically significant trend.</span></p>
+<p><span style="color:#000000;"><strong><span style="text-decoration:underline;"><a href="http://uscigem2012.files.wordpress.com/2012/07/chez_09m.png"><span style="text-decoration:underline;color:#000000;"><img class="aligncenter size-full wp-image-287" title="Screen Shot 2012-10-03 at 7.54.06 PM" src="http://uscigem2012.files.wordpress.com/2012/07/chez_09m.png" alt=""   /></span></a></span></strong></span></p>
 <p><span style="color:#000000;"><strong><span style="text-decoration:underline;"> </span></strong></span></p>
 <p><span style="color:#000000;"><strong>CheY vs. CONTROL                 </strong></span></p>
@@ Line 489: / Line 511: @@
 </tr>
 <tr>
-<td valign="top" width="84"><span style="color:#000000;">Avgerage frequency (Hz)</span></td>
+<td valign="top" width="84"><span style="color:#000000;">Average frequency (Hz)</span></td>
 <td valign="top" width="70">
 <p align="center"><span style="color:#000000;">254</span></p>
@@ Line 545: / Line 567: @@
 </table>
 <p><span style="color:#000000;"><strong><span style="text-decoration:underline;"><a href="http://uscigem2012.files.wordpress.com/2012/10/screen-shot-2012-10-03-at-7-54-06-pm.png"><span style="text-decoration:underline;color:#000000;"><img class="aligncenter size-full wp-image-287" title="Screen Shot 2012-10-03 at 7.54.06 PM" src="http://uscigem2012.files.wordpress.com/2012/10/screen-shot-2012-10-03-at-7-54-06-pm.png?w=960" alt=""   /></span></a></span></strong></span></p>
 <p><span style="color:#000000;"><strong><span style="text-decoration:underline;">CheY vs. CheZ vs. CONTROL</span></strong></span></p>
 <table border="1" cellspacing="0" cellpadding="0">
@@ Line 574: / Line 597: @@
 </tbody>
 </table>
-<p><span style="color:#000000;">In order to better control the tumbling/swimming response in <em>E. coli</em>, we generated biobricks that facilitate the overexpression of <em>cheY</em> and <em>cheZ</em>, which directly influence clockwise/counterclockwise movement of the flagella</span></p>
+<br>
+<p><span style="color:#000000;"></span></p>
 <p><span style="color:#000000;"><strong><span style="text-decoration:underline;">Inducible Tumbling System in cheY and cheZ</span></strong></span></p>
-<p><span style="color:#000000;">In order to test our bacterial communication system, we transformed cheY and cheZ parts into <em>E. coli</em> 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.</span></p>
+<p><span style="color:#000000;">In order to better control the tumbling/swimming response in <em>E. coli</em>, we generated biobricks that facilitate the overexpression of <em>cheY</em> and <em>cheZ</em>, which directly influence clockwise/counterclockwise movement of the flagella.  To test our bacterial communication system, we transformed <em>cheY</em> and <em>cheZ</em> parts into <em>E. coli</em> 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 <em>cheY</em> construct showed tumbling, demonstrating the functionality of the T7 RBS <em>cheY</em> part.</span></p>
 <p><span style="color:#000000;"><a href="http://uscigem2012.files.wordpress.com/2012/10/screen-shot-2012-10-03-at-7-55-07-pm.png"><span style="color:#000000;"><img class="aligncenter size-full wp-image-288" title="Screen Shot 2012-10-03 at 7.55.07 PM" src="http://uscigem2012.files.wordpress.com/2012/10/screen-shot-2012-10-03-at-7-55-07-pm.png?w=960" alt=""   /></span></a></span></p>
 <p><span style="color:#000000;"><a href="http://uscigem2012.files.wordpress.com/2012/10/screen-shot-2012-10-03-at-7-57-32-pm.png"><span style="color:#000000;"><img class="aligncenter size-full wp-image-291" title="Screen Shot 2012-10-03 at 7.57.32 PM" src="http://uscigem2012.files.wordpress.com/2012/10/screen-shot-2012-10-03-at-7-57-32-pm.png?w=960" alt=""   /></span></a></span></p>
 <p><span style="color:#000000;"><strong><span style="text-decoration:underline;">Reconstitution of Flagella</span></strong></span></p>
 <p><span style="color:#000000;">Our team created the following BioBricks that control the assembly and operon regulation of <em>E. coli</em> 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.</span></p>
+<p><span style="color:#000000;"><span style="color:#000000;"><a href="http://uscigem2012.files.wordpress.com/2012/07/biobrick-gene-pathway.gif"><img class="aligncenter" title="BioBrick-Gene-Pathway" src="http://uscigem2012.files.wordpress.com/2012/07/biobrick-gene-pathway.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></span></p>
+<br>
 <p><span style="color:#000000;"><strong><span style="text-decoration:underline;">Future Directions</span></strong></span></p>
-<p><span style="color:#000000;">#5transition text here</span></p>
+<p><span style="color:#000000;">We have begun also characterizing the tumbling response of <em>E. coli</em> 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 <em>cheY</em> and <em>cheZ</em> systems.</span></p>
 <p><span style="color:#000000;"><strong><span style="text-decoration:underline;">pH Experiment</span></strong></span></p>
 <p><span style="color:#000000;"><strong><span style="text-decoration:underline;">CONTROL</span></strong></span></p>