Optimal caffeine concentration on plates - Revision history

Jolim at 18:47, 19 August 2014

2014-08-19T18:47:48Z

Jolim: Added "See also" section

2014-07-03T18:33:51Z

Added "See also" section

Jolim at 13:29, 1 July 2014

2014-07-01T13:29:14Z

Jolim at 13:28, 1 July 2014

2014-07-01T13:28:32Z

Jolim: Protocol updated, data and figure added

2014-07-01T13:26:33Z

Protocol updated, data and figure added

Jolim: Created page

2014-06-19T20:16:58Z

Created page

New page

==Overview==
Before strains with the tetracycline-theophylline fitness module can be grown in broth, we need to know how much caffeine the cells need, to produce an amount of theophylline that grants survival. [http://gcat.davidson.edu/mediawiki-1.19.1/index.php/File:MWSU.pptx Previous experimentation] shows that colonies on plates are able to grow in a ring around caffeine-soaked disks, leading to my hypothesis that there is an <b>optimal range of caffeine</b> in which cells can survive. Too much caffeine or too little caffeine prevents cells from growing. However, we do not know whether cells growing in broth require the same concentration of caffeine as cells growing in plates. This experiment was designed to determine the concentration required by cells growing on plates, specifically pertaining to JM109 strains #12 and #23, [http://gcat.davidson.edu/mediawiki-1.19.1/index.php/File:MWSU.xlsx#file which have been demonstrated] to be the most competent clones on agar plates.

==Protocol==
Prepare 20-mL batches of LB agar with incremental dilutions of caffeine from 20 mM to 40 mM. (This range was guessed based on observation of the distance of colonies from the disk in MWSU photos.)
The agar-caffeine dilutions should have 5 μg/mL tetracycline and 0.5 mg/mL L-arabinose. The negative control should contain no caffeine. The first positive control should have 4 mM theophylline and the second positive control should have no tetracycline. See the below table for details.
Add anhydrous caffeine and anhydrous theophylline to the agar before autoclaving. Add tetracycline and L-arabinose from liquid stocks after autoclaving and cooling to below 60°.

<table class="tableizer-table">
<tr class="tableizer-firstrow"><th>#</th><th>Tetracycline (μg/mL)</th><th>Tet (uL)</th><th>Theophylline (mM)</th><th>Theo (g)</th><th>Caffeine (mM)</th><th>Caff (g)</th><th>L-arabinose (uL)</th><th>Comment</th></tr>
<tr><td>1</td><td>5</td><td>20</td><td>0</td><td>0</td><td>0</td><td>0.000</td><td>40</td><td>Negative control</td></tr>
<tr><td>2</td><td>5</td><td>20</td><td>0</td><td>0</td><td>20</td><td>0.078</td><td>40</td><td> </td></tr>
<tr><td>3</td><td>5</td><td>20</td><td>0</td><td>0</td><td>21</td><td>0.082</td><td>40</td><td> </td></tr>
<tr><td>4</td><td>5</td><td>20</td><td>0</td><td>0</td><td>22</td><td>0.085</td><td>40</td><td> </td></tr>
<tr><td>5</td><td>5</td><td>20</td><td>0</td><td>0</td><td>23</td><td>0.089</td><td>40</td><td> </td></tr>
<tr><td>6</td><td>5</td><td>20</td><td>0</td><td>0</td><td>24</td><td>0.093</td><td>40</td><td> </td></tr>
<tr><td>7</td><td>5</td><td>20</td><td>0</td><td>0</td><td>25</td><td>0.097</td><td>40</td><td> </td></tr>
<tr><td>8</td><td>5</td><td>20</td><td>0</td><td>0</td><td>26</td><td>0.101</td><td>40</td><td> </td></tr>
<tr><td>9</td><td>5</td><td>20</td><td>0</td><td>0</td><td>27</td><td>0.105</td><td>40</td><td> </td></tr>
<tr><td>10</td><td>5</td><td>20</td><td>0</td><td>0</td><td>28</td><td>0.109</td><td>40</td><td> </td></tr>
<tr><td>11</td><td>5</td><td>20</td><td>0</td><td>0</td><td>29</td><td>0.113</td><td>40</td><td> </td></tr>
<tr><td>12</td><td>5</td><td>20</td><td>0</td><td>0</td><td>30</td><td>0.117</td><td>40</td><td> </td></tr>
<tr><td>13</td><td>5</td><td>20</td><td>0</td><td>0</td><td>31</td><td>0.120</td><td>40</td><td> </td></tr>
<tr><td>14</td><td>5</td><td>20</td><td>0</td><td>0</td><td>32</td><td>0.124</td><td>40</td><td> </td></tr>
<tr><td>15</td><td>5</td><td>20</td><td>0</td><td>0</td><td>33</td><td>0.128</td><td>40</td><td> </td></tr>
<tr><td>16</td><td>5</td><td>20</td><td>0</td><td>0</td><td>34</td><td>0.132</td><td>40</td><td> </td></tr>
<tr><td>17</td><td>5</td><td>20</td><td>0</td><td>0</td><td>35</td><td>0.136</td><td>40</td><td> </td></tr>
<tr><td>18</td><td>5</td><td>20</td><td>0</td><td>0</td><td>36</td><td>0.140</td><td>40</td><td> </td></tr>
<tr><td>19</td><td>5</td><td>20</td><td>0</td><td>0</td><td>37</td><td>0.144</td><td>40</td><td> </td></tr>
<tr><td>20</td><td>5</td><td>20</td><td>0</td><td>0</td><td>38</td><td>0.148</td><td>40</td><td> </td></tr>
<tr><td>21</td><td>5</td><td>20</td><td>0</td><td>0</td><td>39</td><td>0.151</td><td>40</td><td> </td></tr>
<tr><td>22</td><td>5</td><td>20</td><td>0</td><td>0</td><td>40</td><td>0.155</td><td>40</td><td> </td></tr>
<tr><td>23</td><td>5</td><td>20</td><td>4</td><td>0.014</td><td>0</td><td>0.000</td><td>40</td><td>Positive control</td></tr>
<tr><td>24</td><td>5</td><td>20</td><td>4</td><td>0.014</td><td>40</td><td>0.155</td><td>40</td><td>Positive control</td></tr>
</table>

Pour each agar dilution into two 35-mm plates, labeled A and B. Flip all plates after 3 hours and let dry for two days.

Incubate LB broth cultures of each clone overnight at 37°, with 0.5 mG/mL L-arabinose and either amp or amp+chlor. Clones with a chaperone require chloramphenicol to prevent curing.

Measure A590 and dilute concentration to 0.05 OD with LB+antibiotic.

For the “A” series, place 2-μL spot of each clone onto the plates.

For the “B” series, spread 10 uL of each clone with 3-5 beads onto the plates. Swirl beads carefully without the lid, discard beads, and replace lid. Store plates inverted.

Incubate all plates at 37° overnight. Let grow for 4 days at RT. Inspect daily for growth. Count colonies per plate or colonies per area to determine the plate with the optimal caffeine concentration.

@@ Line 36: / Line 36: @@
 ==Data==
-[[File:jon-caffeine-growth-in-broth-figure.png|frameless|500px|The number of CFU's is dependent on the concentration of caffeine provided to E. coli transformed with the tetracycline fitness module.]]
+[[File:jon-caffeine-growth-in-agar-figure.png|frameless|500px|The number of CFU's is dependent on the concentration of caffeine provided to E. coli transformed with the tetracycline fitness module.]]
 [[Media:Optimal-caffeine-on-plates-design-data-figure.xlsx]]

@@ Line 36: / Line 36: @@
 ==Data==
-[[File:jon-caffeine-growth-in-broth-figure.png|thumb|500px|left|The number of CFU's is dependent on the concentration of caffeine provided to E. coli transformed with the tetracycline fitness module.]]
+[[File:jon-caffeine-growth-in-broth-figure.png|frameless|500px|The number of CFU's is dependent on the concentration of caffeine provided to E. coli transformed with the tetracycline fitness module.]]
 [[Media:Optimal-caffeine-on-plates-design-data-figure.xlsx]]

@@ Line 5: / Line 5: @@
 Prepare 80-mL batches of LB agar with incremental dilutions of caffeine from 0 mM to 10 mM. (This range was guessed based on a model of caffeine diffusion created by Morgan Spencer.)
 The agar-caffeine dilutions should have 20 μg/mL tetracycline and 0.5 mg/mL L-arabinose. The negative control should contain no caffeine. The first positive control should have 4 mM theophylline and the second positive control should have no tetracycline. See the below table for details.
 Add anhydrous caffeine and anhydrous theophylline to the respective agar batches before autoclaving. Add tetracycline and L-arabinose from liquid stocks after autoclaving and cooling to below 60°.

@@ Line 21: / Line 21: @@
   <tr><td>11</td><td>20</td><td>320</td><td>0</td><td>0</td><td>10</td><td>0.155</td><td>160</td></tr>
   <tr><td>12</td><td>20</td><td>320</td><td>4</td><td>0.058</td><td>0</td><td>0.000</td><td>160</td></tr>
-  <tr><td>13</td><td>20</td><td>320</td><td>4</td><td>0.058</td><td>10</td><td>0.155</td><td>160</td></tr>
+  <tr><td>13</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>160</td></tr>
 </table>

@@ Line 3: / Line 3: @@
 ==Protocol==
-Prepare 20-mL batches of LB agar with incremental dilutions of caffeine from 20 mM to 40 mM. (This range was guessed based on observation of the distance of colonies from the disk in MWSU photos.)
+Prepare 80-mL batches of LB agar with incremental dilutions of caffeine from 0 mM to 10 mM. (This range was guessed based on a model of caffeine diffusion created by Morgan Spencer.)
-The agar-caffeine dilutions should have 5 μg/mL tetracycline and 0.5 mg/mL L-arabinose. The negative control should contain no caffeine. The first positive control should have 4 mM theophylline and the second positive control should have no tetracycline. See the below table for details.
+The agar-caffeine dilutions should have 20 μg/mL tetracycline and 0.5 mg/mL L-arabinose. The negative control should contain no caffeine. The first positive control should have 4 mM theophylline and the second positive control should have no tetracycline. See the below table for details.
-Add anhydrous caffeine and anhydrous theophylline to the agar before autoclaving. Add tetracycline and L-arabinose from liquid stocks after autoclaving and cooling to below 60°.
+Add anhydrous caffeine and anhydrous theophylline to the respective agar batches before autoclaving. Add tetracycline and L-arabinose from liquid stocks after autoclaving and cooling to below 60°.
 <table class="tableizer-table">
-<tr class="tableizer-firstrow"><th>#</th><th>Tetracycline (&#956;g/mL)</th><th>Tet (uL)</th><th>Theophylline (mM)</th><th>Theo (g)</th><th>Caffeine (mM)</th><th>Caff (g)</th><th>L-arabinose (uL)</th><th>Comment</th></tr>
+<tr class="tableizer-firstrow"><th>#</th><th>Tetracycline (&#956;g/mL)</th><th>Tet (uL)</th><th>Theophylline (mM)</th><th>Theo (g)</th><th>Caffeine (mM)</th><th>Caff (g)</th><th>L-arabinose (uL)</th></tr>
-  <tr><td>1</td><td>5</td><td>20</td><td>0</td><td>0</td><td>0</td><td>0.000</td><td>40</td><td>Negative control</td></tr>
+  <tr><td>1</td><td>20</td><td>320</td><td>0</td><td>0</td><td>0</td><td>0.000</td><td>160</td></tr>
-  <tr><td>2</td><td>5</td><td>20</td><td>0</td><td>0</td><td>20</td><td>0.078</td><td>40</td><td>&nbsp;</td></tr>
+  <tr><td>2</td><td>20</td><td>320</td><td>0</td><td>0</td><td>1</td><td>0.016</td><td>160</td></tr>
-  <tr><td>3</td><td>5</td><td>20</td><td>0</td><td>0</td><td>21</td><td>0.082</td><td>40</td><td>&nbsp;</td></tr>
+  <tr><td>3</td><td>20</td><td>320</td><td>0</td><td>0</td><td>2</td><td>0.031</td><td>160</td></tr>
-  <tr><td>4</td><td>5</td><td>20</td><td>0</td><td>0</td><td>22</td><td>0.085</td><td>40</td><td>&nbsp;</td></tr>
+  <tr><td>4</td><td>20</td><td>320</td><td>0</td><td>0</td><td>3</td><td>0.047</td><td>160</td></tr>
-  <tr><td>5</td><td>5</td><td>20</td><td>0</td><td>0</td><td>23</td><td>0.089</td><td>40</td><td>&nbsp;</td></tr>
+  <tr><td>5</td><td>20</td><td>320</td><td>0</td><td>0</td><td>4</td><td>0.062</td><td>160</td></tr>
-  <tr><td>6</td><td>5</td><td>20</td><td>0</td><td>0</td><td>24</td><td>0.093</td><td>40</td><td>&nbsp;</td></tr>
+  <tr><td>6</td><td>20</td><td>320</td><td>0</td><td>0</td><td>5</td><td>0.078</td><td>160</td></tr>
-  <tr><td>7</td><td>5</td><td>20</td><td>0</td><td>0</td><td>25</td><td>0.097</td><td>40</td><td>&nbsp;</td></tr>
+  <tr><td>7</td><td>20</td><td>320</td><td>0</td><td>0</td><td>6</td><td>0.093</td><td>160</td></tr>
-  <tr><td>8</td><td>5</td><td>20</td><td>0</td><td>0</td><td>26</td><td>0.101</td><td>40</td><td>&nbsp;</td></tr>
+  <tr><td>8</td><td>20</td><td>320</td><td>0</td><td>0</td><td>7</td><td>0.109</td><td>160</td></tr>
- <tr><td>9</td><td>5</td><td>20</td><td>0</td><td>0</td><td>27</td><td>0.105</td><td>40</td><td>&nbsp;</td></tr>
+  <tr><td>9</td><td>20</td><td>320</td><td>0</td><td>0</td><td>8</td><td>0.124</td><td>160</td></tr>
- <tr><td>10</td><td>5</td><td>20</td><td>0</td><td>0</td><td>28</td><td>0.109</td><td>40</td><td>&nbsp;</td></tr>
+  <tr><td>10</td><td>20</td><td>320</td><td>0</td><td>0</td><td>9</td><td>0.140</td><td>160</td></tr>
-  <tr><td>11</td><td>5</td><td>20</td><td>0</td><td>0</td><td>29</td><td>0.113</td><td>40</td><td>&nbsp;</td></tr>
+  <tr><td>11</td><td>20</td><td>320</td><td>0</td><td>0</td><td>10</td><td>0.155</td><td>160</td></tr>
- <tr><td>12</td><td>5</td><td>20</td><td>0</td><td>0</td><td>30</td><td>0.117</td><td>40</td><td>&nbsp;</td></tr>
+  <tr><td>12</td><td>20</td><td>320</td><td>4</td><td>0.058</td><td>0</td><td>0.000</td><td>160</td></tr>
- <tr><td>13</td><td>5</td><td>20</td><td>0</td><td>0</td><td>31</td><td>0.120</td><td>40</td><td>&nbsp;</td></tr>
+  <tr><td>13</td><td>20</td><td>320</td><td>4</td><td>0.058</td><td>10</td><td>0.155</td><td>160</td></tr>
 </table>
-Pour each agar dilution into two 35-mm plates, labeled A and B. Flip all plates after 3 hours and let dry for two days.
+Measure A590. Combine the clones and dilute with LB broth so that each is at 0.025 OD.
-Incubate LB broth cultures of each clone overnight at 37°, with 0.5 mG/mL L-arabinose and either amp or amp+chlor. Clones with a chaperone require chloramphenicol to prevent curing.
+Incubate all plates at 37° overnight. Let grow for 2 days at RT. Inspect daily for growth.
-Measure A590 and dilute concentration to 0.05 OD with LB+antibiotic.
+==Data==
-For the “A” series, place 2-μL spot of each clone onto the plates.
+[[File:jon-caffeine-growth-in-broth-figure.png|thumb|500px|left|The number of CFU's is dependent on the concentration of caffeine provided to E. coli transformed with the tetracycline fitness module.]]
+[[Media:Optimal-caffeine-on-plates-design-data-figure.xlsx]]