Team:UIUC Illinois/Notebook

From 2014.igem.org

(Difference between revisions)
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cdh PCR redo:
cdh PCR redo:
-
<ul style="text-indent:30px;">
+
Made 4 master mixes: 5uL gDNA CBB1, 45uL DDH2O
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<li>Made 4 master mixes: 5uL gDNA CBB1, 45uL DDH2O</li>
+
-
</ul><br><br>
+
Master Mix 1: Regular
Master Mix 1: Regular
<ul style="text-indent:30px;">
<ul style="text-indent:30px;">
-
<li>20uL Buffer G</li>
+
20uL Buffer G
-
<li>4uL forward primer</li>
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4uL forward primer
-
<li>4uL reverse primer</li>
+
4uL reverse primer
-
<li>0.4uL Q5 polymerase</li>
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0.4uL Q5 polymerase
-
<li>2uL template</li>
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2uL template
-
<li>9.6uL H2O</li>
+
9.6uL H2O
-
</ul><br><br>
+
 
-
Master Mix 2: Forward primer
+
-
<ul style="text-indent:30px;">
+
-
<li>20uL Buffer G</li>
+
-
<li>8uL forward primer</li>
+
-
<li>0uL reverse primer</li>
+
-
<li>0.4uL Q% polymerase</li>
+
-
<li>2uL template</li>
+
-
<li>9.6uL H2O</li>
+
-
</ul><br><br>
+
-
Master Mix 3: Reverse primer
+
-
<ul style="text-indent:30px;">
+
-
<li>20uL Buffer G</li>
+
-
<li>0uL forward primer</li>
+
-
<li>8uL reverse primer</li>
+
-
<li>0.4uL Q5</li>
+
-
<li>2uL template</li>
+
-
<li>9.6uL H2O</li>
+
-
</ul><br><br>
+
-
Master Mix 4: gDNA (10x)
+
-
<ul style="text-indent:30px;">
+
-
<li>20uL Buffer G</li>
+
-
<li>4uL forward primer</li>
+
-
<li>4uL reverse primer</li>
+
-
<li>0.4uL Q5 polymerase</li>
+
-
<li>2uL template</li>
+
-
<li>9.6uL H2O</li>
+
-
</ul><br><br>
+
-
PCR Procedure: Made 4 10uL samples of each master mix
+
-
Set up thermocycler with the following settings:
+
-
<ul style="text-indent:30px;">
+
-
<li>98C: 30 min</li>
+
-
<li>40x cycles that ended up being:</li>
+
-
<li>98C for 20 minutes</li>
+
-
<li>Gradient temperature for 20 minutes: 44.9C, 50.8C. 58.7C, 65.1C</li>
+
-
<li>72C for 80 min</li>
+
-
<li>72C for 5 minutes</li>
+
-
<li>Paused at 4C</li>
+
-
</ul><br><br>
+
-
Optimized shuttle vector growth environments: From 1 mg/mL concentrate
+
-
<ul style="text-indent:30px;">
+
-
<li>1mL of each concentration into 2 falcon tubes</li>
+
-
<li>Inoculated 3mL LB with cells + Erm stocks</li>
+
-
<li>Also had 0ug/mL as a negative control</li>
+
-
<li>8 tubes in total</li>
+
-
</ul><br><br>
+
-
<table width="100%" border="1px solid black" >
+
-
<tr>
+
-
<th>Concentrations(ug/mL)</th>
+
-
<th>H2O(mL)</th>
+
-
<th>Erm(mL)</th>
+
-
</tr>
+
-
<tr>
+
-
<td>50</td>
+
-
<td>3.8</td>
+
-
<td>0.2</td>
+
-
</tr>
+
-
<tr>
+
-
<td>25</td>
+
-
<td>1.5</td>
+
-
<td>1.5</td>
+
-
</tr>
+
-
<tr>
+
-
<td>10</td>
+
-
<td>0.5</td>
+
-
<td>0.5</td>
+
-
</tr>
+
-
</table>
+
-
Inoculating Optogene Liquid Cultures:
+
-
<ul style="text-indent:30px;">
+
-
<li>Added 10mL liquid LB and 10uL chloramphenicol to each of the four falcon tubes</li>
+
-
<li>Picked colonies off each labeled plate and added to tubes as follows:
+
-
<ul style="text-indent:30px;">
+
-
<li>Plate
+
-
<ul style="text-indent:30px;">
+
-
<li>Green light 1</li>
+
-
<li>Green light 2</li>
+
-
<li>Red light sensor 1</li>
+
-
<li>Red light sensor 2</li>
+
-
</ul>
+
-
</ul>
+
-
<li>Incubated all 4 tubes in 37C shaker at 5:15pm</li>
+
-
</ul><br><br>
+
-
Inoculated E. coli guaB pdCAF (I4) in 3uL LB + 3uL CM<br><br>
+
-
Inoculated Top 10 pdCAF (I5) in 3uL LB + 3uL CM<br><br>
+
-
Placed in 37C shaker at 5:15pm<br><br>
+
</div>
</div>
</html>
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</html>
</html>
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<html><div id="Monday_4st_August.html"></div></html>
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<html><div id="Monday_4st_July.html"></div></html>
<html>
<html>
<div class ="tbnote">
<div class ="tbnote">
-
<h2>Monday 4<sup>st</sup> August</h2>
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<h2>Monday 4<sup>th</sup> July</h2>
<a href="https://2014.igem.org/Team:UIUC_Illinois/Project/Overview" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>
<a href="https://2014.igem.org/Team:UIUC_Illinois/Project/Overview" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>
<div style="clear: both;"></div>
<div style="clear: both;"></div>
-
 
M9 Caffeine Media
M9 Caffeine Media
<ul style="text-indent:30px;">
<ul style="text-indent:30px;">
 +
<li>200uM 200mL caffeine</li>
<li>200uM 200mL caffeine</li>
<li>50 M9 5x salts</li>
<li>50 M9 5x salts</li>
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<br><br>
<br><br>
M9-Theobromine Media
M9-Theobromine Media
-
 
<li>200mL 200uM Theobromine</li>
<li>200mL 200uM Theobromine</li>
<li>50mL M9 5x salts</li>
<li>50mL M9 5x salts</li>
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<li>1 hour 37C incubation</li>
<li>1 hour 37C incubation</li>
<li>20 min 80C heat inactivation</li>
<li>20 min 80C heat inactivation</li>
-
 
</ul>
</ul>
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</html>
</html>
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<html><div id="Tuesday_5th_August.html"></div></html>
 
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<html>
 
-
<div class ="tbnote">
 
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<h2>Tuesday 5<sup>th</sup> August</h2>
 
-
<a href="https://2014.igem.org/Team:UIUC_Illinois/Project/Overview" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>
 
-
<div style="clear: both;"></div>
 
-
             
 
-
Ran gel of pdCAF3 digestion
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<html><div id="Monday_11th_August.html"></div></html>
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<br><br>
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-
Set up feeding experiment cultures
+
-
<ul style="text-indent:30px;">
+
-
<li>Added 50uL caffeine M9/theobromine M9 to 250mL flasks</li>
+
-
<li>Added 50uL of CM34 to 4 of 6 flasks</li>
+
-
<li>Added 500uL of cells to flasks</li>
+
-
<li>Took 1mL of each culture as zero time point, placed in -20C</li>
+
-
<li>Placed flasks in 30C shaker</li>
+
-
 
+
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+
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</ul>
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</div>
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</html>
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<html><div id="Wednesday_6th_August.html"></div></html>
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<html>
<html>
<div class ="tbnote">
<div class ="tbnote">
-
<h2>Wednesday 6<sup>th</sup> August</h2>
+
<h2>Monday 11<sup>th</sup> August</h2>
<a href="https://2014.igem.org/Team:UIUC_Illinois/Project/Overview" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>
<a href="https://2014.igem.org/Team:UIUC_Illinois/Project/Overview" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>
<div style="clear: both;"></div>
<div style="clear: both;"></div>
-
             
 
<ul style="text-indent:30px;">
<ul style="text-indent:30px;">
-
Digsted pdCAF3 using XhoI
+
Resuspended primers in water
-
<li>5uL cutsmart buffer</li>
+
<li>Spin down for 5 seconds</li>
-
<li>.5uL XhoI</li>
+
<li>Inverted several times, set for 3 minutes</li>
-
<li>7.52uL pdCAF3</li>
+
<li>Inverte again, let sit for 3 more minutes</li>
-
<li>36.98uL H2O</li>
+
<li>Invert before use</li>
-
<li>1 hour 37C incubation</li>
+
-
<li>Ran gel</li>
+
<br><br>
<br><br>
-
Took 24 hour time point for feeding experiment, stored in -20
+
PCR
 +
<li>57uL H2O + 1.6uL dNTP + 16uL buffer + 0.8uL polymerase in tube 1</li>
 +
<li>9uL of 1 + 0.25uL BB primer for + 0.25uL primer rev + 0.5uL template (PSBC13) in tube 8</li>
 +
<li>Add 0.5uL pdCAF3 to tube 1</li>
 +
<li>9uL of 1 to tube 2-7, 0.25 rev + 0.25 for to each of the tubes respectively</li>
 +
<li>Spin down for 5 seconds</li>
 +
<li>Put tube 2-8 in PCR machine</li>
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<html><div id="Thursday_7th_August.html"></div></html>
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<html><div id="Tuesday_12th_August.html"></div></html>
<html>
<html>
<div class ="tbnote">
<div class ="tbnote">
-
<h2>Thursday 7<sup>th</sup> August</h2>
+
<h2>Tuesday 12<sup>th</sup> August</h2>
<a href="https://2014.igem.org/Team:UIUC_Illinois/Project/Overview" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>
<a href="https://2014.igem.org/Team:UIUC_Illinois/Project/Overview" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>
<div style="clear: both;"></div>
<div style="clear: both;"></div>
-
             
 
-
Took 48 hour time points for feeding experiments, stored in -20C
 
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</ul>
 
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</div>
 
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</html>
 
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<html><div id="Friday_8th_August.html"></div></html>
 
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<html>
 
-
<div class ="tbnote">
 
-
<h2>Friday 8<sup>th</sup> August</h2>
 
-
<a href="https://2014.igem.org/Team:UIUC_Illinois/Project/Overview" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>
 
-
<div style="clear: both;"></div>
 
-
             
 
<ul style="text-indent:30px;">
<ul style="text-indent:30px;">
-
Made 250uL 100uM (7.5pH) KPi buffer
+
456uL H2O, 128uL buffer, 12.8uL dNTP, 6.4uL polymerase into tube A
-
<li>20.75uL of 1M K2HPO4
+
-
<li>4.25uL of 1M KH2PO4
+
-
<li>2.25uL of H2O
+
<br><br>
<br><br>
-
Centrifuged, washed experimental cultures of IG4 with KPi buffer
+
9uL of A + 0.25uL BB for + 0.25uL BB rev + 0.5uL purified green in tube 8
<br><br>
<br><br>
-
Resuspended in 25uL KPi buffer
+
0.5uL pdCAF to tube A
<br><br>
<br><br>
-
Made 10% casamino acid solution
+
72uL A to tube 1-6, 9uL A to tube 7, 0.25 BB rev/for to 7, 2uL for/2uL rev to 1-6 respectively
-
<li>5g casamino acids + 50mL H2O</li>
+
<br><br>
-
 
+
Spin down for 5 seconds
 +
<br><br>
 +
PCR in cycle 1
 +
<table width="100%" border="1px solid black" >
 +
<tr>
 +
<th>Tube</th>
 +
<th>Primers</th>
 +
<th>Template</th>
 +
<th>Volume(uL)</th>
 +
 +
</tr>
 +
        <tr>
 +
<td>1</td>
 +
<td>ndmA 1</td>
 +
<td>pdCAF</td>
 +
<td>80</td>
 +
 +
</tr>
 +
  <tr>
 +
<td>2</td>
 +
<td>ndmA 2</td>
 +
<td>pdCAF</td>
 +
<td>80</td>
 +
 +
</tr>
 +
  <tr>
 +
<td>3</td>
 +
<td>ndmB 1</td>
 +
<td>pdCAF</td>
 +
<td>80</td>
 +
 +
</tr>
 +
  <tr>
 +
<td>4</td>
 +
<td>ndmB 2</td>
 +
<td>pdCAF</td>
 +
<td>80</td>
 +
 +
</tr>
 +
  <tr>
 +
<td>5</td>
 +
<td>ndmC</td>
 +
<td>pdCAF</td>
 +
<td>80</td>
 +
 +
</tr>
 +
  <tr>
 +
<td>6</td>
 +
<td>ndmD + gst9</td>
 +
<td>pdCAF</td>
 +
<td>80</td>
 +
 +
</tr>
 +
  <tr>
 +
<td>7</td>
 +
<td>BB</td>
 +
<td>pdCAF</td>
 +
<td>10</td>
 +
 +
</tr>
 +
  <tr>
 +
<td>8</td>
 +
<td>BB</td>
 +
<td>Green Optogene</td>
 +
<td>10</td>
 +
 +
</tr>
 +
</table>
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<!--/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////-->
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Revision as of 23:08, 17 October 2014


Monday 9th June

Made LB broth using 10 grams of bacto agar + 12 grams HiVeg LB broth + 600 mL ddH2O

Autoclaved 1.5 mL Eppendorf tubes as well as LB

Poured plates of LB and chloramphenicol (CAM)

Streaked 2 plates with E. coli DguaB (pDCAF3)

Incubated in 37 degrees celsius

Aliquoted kanamycin (KAN) in four 1mL tubes

Tuesday 10th June

Autoclaved at 1:30pm

  • 0.8 grams of YNB + 156 mL H2O
  • 28 grams of salts + 490 mL H2O

Used sterile filter for 1M MgSO4 and 1M CaCl2

Suspended 5 uL of CM34 (antibiotic) + 5 mL of LB + 1 colony of pDCAF3 E. coli

Added .5 grams of caffeine + 400 uL of 1M MgSO4 + 20 uL 1M CaCl2 + 40 mL M95x salts + H2O to YNB + H2O mixture

  • Labeled this "M9 media"

Inoculated frozen stock of E. coli guaB pDCAF3 in 5 mL of M9 media

Placed sample in 30 degree celsius shaker for culturing

Wednesday 11th June

Inoculated CBB1 frozen stock in 5 mL of M9 media

Placed sample in 30 degree celsius shaker

Purified pDCAF plasmid using DNA mini kit

  • Plasmid concentration was 132.9 ng/uL


Friday 13th June

Added 10 uL deionized H2O into the J23114 promoter plasmid (Ampᴿ)

Followed bacterial transformation protocol to prepare/grow culture overnight

Inoculated 5 mL of M9 with CBB1 and placed it in 30 degree celsius shaker for 3 days

  • Previous CBB1 culture did not grow, showed slight growth
  • pDCAF3 strain in M9 did grow in the 30 degree celsius shaker


Monday 16th June

No growth in M9 by CBB1

To grow the promoter plasmid:

  • 5 mL of LB + 1 uL Amp
  • Label tubes
  • Grow in 30 degree celsius shaker overnight
New CBB1 trials:

  • 2 tubes of 1 mL M9 + 2 tubes of 1 mL LB each with 10 uL of sample


Purify genomic DNA plasmid following the Wizard Protocol

  • Added lysis buffer before centrifuging. So we separated (translate) sample into 2 tubes and will (translate) accordingly
  • We put our 2 promoter plasmid plates in the 4 degree celsius shelf


Tuesday 17th June

Quantified gDNA with TECAN
  • Result: 5.4 ng/uL


Made 2 plates of M9 culture and 2 plates of CBB1 culture (100 uL used) then placed them in 37 degree celsius shaker

Wednesday 18th June

Autoclaved 50% glycerol

Made four 25% glycerol stock solutions (stored in the -80 degree celsius freezer):

  • CBB1 in M9 (x2 1.5 mL tubes)
  • Promoter 1
  • Promoter 2 (both promoter plasmids are J23114 Ampᴿ)
Purified both promoter plasmids using the purification protocol
  • Stored in the -20 degree celsius freezer in the DNA box


Thursday 19th June

Quantified promoter plasmid (J23114 #1 and #2)
    #1: 342.9 ng/ul with a ratio of 1.9 #2: 373.2 ng/ul with a ratio of 1.91


Checking to see if expired enzymes work: Using the J23114 promoter plasmid, we will see if the 3-year expired SpeI works
  • We combined 1 uL of 2.1 buffer, 1 uL of Spe1, and 6 uL of J23114 promoter
  • Heat bath at 37 degrees celsius for 1 hour because 2.1 buffer is <100% effective on this enzyme
  • Then we will run gel electrophoresis: Concluded that the enzyme works


Cutting the promoter with restriction enzymes
  • Combined 24 uL of J23114 promoter + 3 uL of CutSmart buffer + 1 uL of each EcoRI and SpeI
  • Heat bath at 37 degrees celsius for 1 hour + 20 minute heat-kill at 80 degrees celsius


Cutting linearized backbone
  • Linearized backbone with chloraphenicol marker (12 uL) + 12 uL water + 1 uL of each EcoRI, PstI, and DpnI + 3 uL 2.1 buffer
  • Heat bath at 37 degrees celsius for 1 hour + 20 minute heat-kill at 80 degrees celsius


Wednesday 9th July

Made 400mL of M9 solution
  • Made 40% glucose solution of 50mL: 2g glucose
  • Added the following to 316mL of DI H2O: Na2HPO4: 13.56g Na2HPO4, 2g NH4Cl, 1g NaCl, 6g KH2PO4
  • Added 80mL 5x M9 salt solution (20% of 400mL)
  • Added 800uL MgSO4 + 40uL CaCl2
  • Added 4mL glucose solution using syringe
  • Stirred until all dissolved


Removed competent cells from the -80 freezer

Thawed on ice for 30 minutes

Removed CAM agar plate from 4 degree cold room

Mixed 4uL of pDCAF3 plasmid with 30uL competent cells in microcentrifuge tube, placed on ice for 30 minutes

Placed mixture in water bath (42C) for 45 seconds

Placed mixture on ice for 2 minutes

Added 300uL LB to mixture

Placed in 37 degree shaker for 45 minutes

Plated cells on CAM plate, placed in 37 degree drawer overnight

Thursday 10th July

GuaB/pDCAF suspension in caffeine/theobromine
  • 0.0975g/10mL caffeine, 0.09g/10mL -> 50 mM
  • 2.5mL of M9 (with Glucose) + 2.4875mL of H2O + 12.5uL of caffeine/theobromine
  • Use syringe to filter out 10mL of caffeine/theobromine into new conical test tubes
  • Take out guaB/pDCAF from -80C freezer, suspend it using 10uL pipette tips
  • Place them into 37C inclubator overnight


Tuesday 15th July

Retrieved lactobacillus WCFS from Dr. Lu

Wednesday 16th July

Incubate 4 tubes with LB+CM
  • Red 1 and 2
  • Green 1 and 2
  • Placed in shaker at 12:20pm


Thursday 17th July

Made 1mg/mL Erm of 50mL

Used syringe to filter out

25mL Erm into LB agar

Made 13 places

Erm is in -20C

Friday 18th July

Streak out Erm-R onto Erm plates (made 2)

Put them into incubator (start: 9:35am)

Monday 21th July

cdh PCR redo: Made 4 master mixes: 5uL gDNA CBB1, 45uL DDH2O Master Mix 1: Regular
    20uL Buffer G 4uL forward primer 4uL reverse primer 0.4uL Q5 polymerase 2uL template 9.6uL H2O

Saturday 26th July

MRS agar of 200mL
  • 3g agar (15g/L)
  • 10.41g MRS


      • 300mL LB + 3uL CM34 + IG4

      300mL LB + 3uL CM34 + IG5

Monday 28th July

Diluted caffeine and theobromine solutions for HPLC
  • 500uM Caffeine (10mL)
  • 500uM Theobromine (10mL)
  • 100uM Caffeine (10mL)
  • 100uM Theobromine (10mL)
  • 250uM Caffeine + 250uM Theobromine (10mL)


(page 169 on bottom, can’t read XW’s part)
  • Take 1mL of I4/I5 LB cultures and spin for 2 minutes at 8000 rpm
  • Removed supernatant
  • Added 1mL of OH salt, resuspended
  • 2 minutes at 8000 rpm
  • Removed supernatant
  • Resuspended in 1mL of M9 media

Wednesday 30th July

Ran gDNA extraction
  • Quantification: 1.8 ratio

Thursday 31th July

gDNA qualtify: 97.5 ng/mL, 1.76 ratio

cdh PCR
  • 30uL H20
  • 10uL Phusion HF
  • 1uL dNTP 10x
  • 2.5uL rev.
  • 2.5uL for.
  • 3.5uL template
  • 0.5uL polymerase
  • Made 4 10uL samples from master mix
  • Gradient: 44.9, 50.8, 58.9, 65.1C
  • Cycle 3 was used


    • Performed restriction digestion of pdCAF3 and green optogene
  • pdCAF3
    • .5uL Cutsmart buffer
    • .5uL Saci
    • .5uL EcoRI-HF
    • 7.52uL pdCAF3
    • 36.48uL H2O
  • Green
    • .5uL Cutsmart buffer .5uL NcoI .5uL NheI 8.4uL Green optogene 35.1 uL H2O
  • Incubated at 37C waterbath for 1 minute
  • Gel run at 120V for 25 minutes with a 1kb ladder


    • Cultured IG4/IG5 in M9 and no CM
  • 3mL M9, 300uL Caffeine, for only IG4, 30uL cells
  • Placed in 37 degree shaker

Monday 4th July

M9 Caffeine Media
  • 200uM 200mL caffeine
  • 50 M9 5x salts
  • .5mL MgSO4
  • .025mL CaCl2
  • 1.25mL 40% glucose


    • M9-Theobromine Media
  • 200mL 200uM Theobromine
  • 50mL M9 5x salts
  • .5mL MgSO4
  • .025mL CaCl2
  • 1.25mL 40% glucose


    • pdCAF3 Digestion
  • 5uL cutsmart buffer
  • 1uL PstI
  • .5uL XhoI
  • 7.52uL pdCAF3
  • 35.98uL H2O
  • 1 hour 37C incubation
  • 20 min 80C heat inactivation


Monday 11th August

    Resuspended primers in water
  • Spin down for 5 seconds
  • Inverted several times, set for 3 minutes
  • Inverte again, let sit for 3 more minutes
  • Invert before use


    • PCR
  • 57uL H2O + 1.6uL dNTP + 16uL buffer + 0.8uL polymerase in tube 1
  • 9uL of 1 + 0.25uL BB primer for + 0.25uL primer rev + 0.5uL template (PSBC13) in tube 8
  • Add 0.5uL pdCAF3 to tube 1
  • 9uL of 1 to tube 2-7, 0.25 rev + 0.25 for to each of the tubes respectively
  • Spin down for 5 seconds
  • Put tube 2-8 in PCR machine


Tuesday 12th August

    456uL H2O, 128uL buffer, 12.8uL dNTP, 6.4uL polymerase into tube A

    9uL of A + 0.25uL BB for + 0.25uL BB rev + 0.5uL purified green in tube 8

    0.5uL pdCAF to tube A

    72uL A to tube 1-6, 9uL A to tube 7, 0.25 BB rev/for to 7, 2uL for/2uL rev to 1-6 respectively

    Spin down for 5 seconds

    PCR in cycle 1
    Tube Primers Template Volume(uL)
    1 ndmA 1 pdCAF 80
    2 ndmA 2 pdCAF 80
    3 ndmB 1 pdCAF 80
    4 ndmB 2 pdCAF 80
    5 ndmC pdCAF 80
    6 ndmD + gst9 pdCAF 80
    7 BB pdCAF 10
    8 BB Green Optogene 10


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