Team:Lethbridge Canada/Notebook

From 2012hs.igem.org

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(Information Sheet)
(June 16, 2012: PCR of J23100-Mlc-J06702 with Prefix and Suffix (Antisense))
 
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<i> Brainstorming Project Ideas</i>
<i> Brainstorming Project Ideas</i>
* <b>Pests</b>  
* <b>Pests</b>  
-
**Bacterial Pest attractor (engineer bacteria to produce a substance, such as a pheromone or smell, that attracts and kills pests such as insects)  
+
**Bacterial Pest attractor (engineer bacteria to produce a substance, such as a pheromone or smell, that attracts and kills pests such as insects).
-
**Natural pesticides (engineer bacteria to produce a substance that repels or kills pests, such as those that harm crops, that can later be implemented into plants) <br> <br>
+
**Natural pesticides (engineer bacteria to produce a substance that repels or kills pests, such as those that harm crops, that can later be implemented into plants). <br> <br>
* <b>Water/Environment</b>  
* <b>Water/Environment</b>  
-
**Desalination of water (engineering bacteria to get rid of salt in salt water in order to make drinkable water from sea water)  
+
**Desalination of water (engineering bacteria to get rid of salt in salt water in order to make drinkable water from sea water).
**Getting rid of estrogen mimicking compounds in water (engineering bacteria to degrade them or sequester them)  
**Getting rid of estrogen mimicking compounds in water (engineering bacteria to degrade them or sequester them)  
-
**Waste treatment (engineering bacteria that can be integrated into waste water treatment)
+
**Waste treatment (engineering bacteria that can be integrated into waste water treatment).
-
**CFCs (engineer bacteria to produce metabolites that break down chlorofluorocarbons—compounds that contribute to the degradation of the ozone layer)<br><br>  
+
**CFCs (engineer bacteria to produce metabolites that break down chlorofluorocarbons—compounds that contribute to the degradation of the ozone layer).<br><br>  
* <b>Health </b>
* <b>Health </b>
-
**Stomach ulcers (creating a medication containing engineered bacteria to specifically target and kill Helicobacter pylori – the organism that causes stomach ulcers)  
+
**Stomach ulcers (creating a medication containing engineered bacteria to specifically target and kill Helicobacter pylori – the organism that causes stomach ulcers).
-
**Diabetes (engineered bacteria as Islet cells to produce insulin)  
+
**Diabetes (engineered bacteria as Islet cells to produce insulin).
-
**Allergies/Immune system (engineer bacteria to produce antihistamines or alter epitopes)  
+
**Allergies/Immune system (engineer bacteria to produce antihistamines or alter epitopes).
-
**Bone density (engineer bacteria to produce and secrete calcium and other compounds to help heal broken bones or to prevent osteoporosis) <br><br>
+
**Bone density (engineer bacteria to produce and secrete calcium and other compounds to help heal broken bones or to prevent osteoporosis). <br><br>
* <b>Process Improvement </b>
* <b>Process Improvement </b>
-
**Oil fractionation catalyst  (engineering bacteria to improve the separation of crude oil into valuable fraction and waste fraction)
+
**Oil fractionation catalyst  (engineering bacteria to improve the separation of crude oil into valuable fraction and waste fraction).
-
**Nitrate fixation (engineer bacteria to improve nitrogen fixation so not as much fertilizer is needed) <br><br>
+
**Nitrate fixation (engineer bacteria to improve nitrogen fixation so not as much fertilizer is needed). <br><br>
* <b>Kill Switches </b>  
* <b>Kill Switches </b>  
-
**Engineering bacteria to undergo induced or programmed cell death in order to control the organism) <br><br>
+
**Engineering bacteria to undergo induced or programmed cell death in order to control the organism). <br><br>
===The chosen project===
===The chosen project===
-
*After dividing in to a few groups based on which project was preferred, each group made a presentation on the proposed project. In the end, we decided to go with the glucose sensing project.<br><br>
+
*After dividing in to a few groups based on which project was preferred, each group made a presentation on the proposed project. In the end, we decided to go with the '''glucose sensing project'''.<br><br>
===Project outline===
===Project outline===
-
<i>Possible things to consider</i>
+
<i>Possible things to consider:</i>
* <b>Glucose and insulin </b>
* <b>Glucose and insulin </b>
Line 70: Line 70:
* <b>Kill switch</b>
* <b>Kill switch</b>
-
** Control and regulation <br><br>
 
* <b>Delivery system </b>
* <b>Delivery system </b>
**Intravenous
**Intravenous
**NASA Biocapsule <br>
**NASA Biocapsule <br>
-
 
==April==
==April==
Line 84: Line 82:
'''Wiki and Mascot Design'''<br>
'''Wiki and Mascot Design'''<br>
-
<Blockquote>''This group is responsible to obtaining and uploading all necessary information to the wiki. This includes team pictures and biographies, experiments and results, and the projects of the other sub-groups. This will be a priority over mascot design''
+
<Blockquote>''This group is responsible to obtaining and uploading all necessary information to the wiki. This includes team pictures and biographies, experiments and results, and the projects of the other sub-groups. This will be a priority over mascot design.''
<Blockquote>Leader: Erin Kelly<br>
<Blockquote>Leader: Erin Kelly<br>
1. Erin Kwan<br>
1. Erin Kwan<br>
Line 100: Line 98:
'''Biosafety'''<br>
'''Biosafety'''<br>
-
<Blockquote>''This group must document the safety protocols that the Lethbridge team practices in the lab''  
+
<Blockquote>''This group must document the safety protocols that the Lethbridge team practices in the lab.''  
<Blockquote>Leader: Marissa Guzzi<br>
<Blockquote>Leader: Marissa Guzzi<br>
1. Branden Black<br>
1. Branden Black<br>
Line 133: Line 131:
Transformation of parts from kit plate:
Transformation of parts from kit plate:
-
 
*B0034 in pSB1A2 <br>
*B0034 in pSB1A2 <br>
Line 140: Line 137:
*E1010 in pSB2K3 <br>
*E1010 in pSB2K3 <br>
-
Overnight cultures were also made of:
 
-
*pMA-T K33109 (HST-1)<br>
+
2µL of DNA was added to 20µL DH5ɑ cells
-
*pSB1AK3 B0014<br>
+
 +
*Incubated on ice for 30 mins<br>
 +
*Cells were then heat shocked for 45sec in water bath at 42 degrees Celsius<br>
 +
*Immediately after, cells were incubated on ice for 5 mins<br>
 +
*400µL SOC media was added and cells were resuspended<br>
 +
*Cells were then placed in shaker for 1h @ 37 degrees Celsius<br>
 +
 +
*200µL of this culture was plated on AMP plates<br>
 +
 +
*Plates were incubated at 37 degrees for appx. 16hrs.<br>
 +
 +
 +
Overnight cultures were also made of:
 +
 +
*pMA-T K33109 (HST-1)<br>
 +
*pSB1AK3 B0014<br>
==='''May 9, 2012: Miniprep and Restriction of Parts'''===
==='''May 9, 2012: Miniprep and Restriction of Parts'''===
Line 198: Line 208:
*K93005 pSB1A2 <br>
*K93005 pSB1A2 <br>
*B0034 pSB1A2<br>
*B0034 pSB1A2<br>
 +
 +
==='''May 10, 2012: Miniprep and Restriction of Parts'''===
==='''May 10, 2012: Miniprep and Restriction of Parts'''===
Line 203: Line 215:
Overnight cultures from May 9th were miniprepped and restricted in order to determine sizes
Overnight cultures from May 9th were miniprepped and restricted in order to determine sizes
-
[[Image:May_10th_restriction.JPG|left|637px]]
+
[[Image:May_10th_restriction_tubes.JPG‎ |left|640px]]
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
Line 226: Line 251:
Restrictions were incubated at 37˚C for 30min then stored in -20˚C.
Restrictions were incubated at 37˚C for 30min then stored in -20˚C.
 +
 +
==='''May 11th: Electrophoresis of restricted parts from May 10th'''===  
==='''May 11th: Electrophoresis of restricted parts from May 10th'''===  
Line 265: Line 292:
-
 
+
*We also grew up J04500 in pSB1AK3 for assembly
-
 
+
-
We also grew up J04500 in pSB1AK3 for assembly
+
Line 275: Line 300:
(adding extra bp to get into fusion standard)
(adding extra bp to get into fusion standard)
-
Add 17.8 uL to 8 tubes with: <br>
+
Add 17.8 µL to 8 tubes with: <br>
-
*Pfu polymerase 0.2 uL <br>
+
*Pfu polymerase 0.2 µL <br>
-
*Template DNA 2 uL<br>
+
*Template DNA 2 µL<br>
*Put tubes on gradient function program Pfu test with various annealing temperatures:
*Put tubes on gradient function program Pfu test with various annealing temperatures:
Line 319: Line 344:
*Ligated together E1010 fusion & B0015  
*Ligated together E1010 fusion & B0015  
-
 
-
 
-
 
-
 
-
 
Line 332: Line 352:
Transformation:
Transformation:
-
2uL of each ligation mix was added to 20uL DH5ɑ cells, then incubated on ice for 30 mins<br>
+
2µL of each ligation mix was added to 20µL DH5ɑ cells
 +
 
 +
*Incubated on ice for 30 mins<br>
*Cells were then heat shocked for 45sec in water bath at 42 degrees Celsius<br>
*Cells were then heat shocked for 45sec in water bath at 42 degrees Celsius<br>
-
*Immediately after, cells were incubated on ice 5 mins<br>
 
-
 
-
*400uL SOC media was added and cells were resuspended<br>
 
-
 
-
*Cells were then placed in shaker for 1h @ 37 degrees<br>
 
-
 
-
*200uL of this culture was plated on AMp plates<br>
 
-
 
-
*Plates were incubated at 37 degrees for appx. 16h<br>
 
 +
*Immediately after, cells were incubated on ice for 5 mins<br>
 +
*400µL SOC media was added and cells were resuspended<br>
 +
*Cells were then placed in the shaker for 1h @ 37 degrees<br>
 +
*200µL of this culture was plated on AMP plates<br>
 +
*Plates were incubated at 37 degrees for appx. 16hrs.<br>
==='''May 14th: Picking Cells from May 13th transformation'''===  
==='''May 14th: Picking Cells from May 13th transformation'''===  
Line 356: Line 374:
*Picked cells were put into 5ml LB media with kanamycin  
*Picked cells were put into 5ml LB media with kanamycin  
-
Cells did not grow
+
*Cells did not grow
-
 
+
-
 
+
-
 
+
Line 400: Line 415:
-
*Add 49.5uL to 4 PCR tubes and add 0.5uL Taq polymerase and 1uL template DNA to each<br>
+
*Add 49.5µL to 4 PCR tubes and add 0.5µL Taq polymerase and 1µL template DNA to each<br>
-
*E1010 (fusion) primer annealing temp 55.5˚C
+
*E1010 (fusion) primer  
 +
*The annealing temp was 55.5˚C
Line 439: Line 455:
-
*Add 49.5 uL of MM to 4 PCR tubes and add 0.5 uL Taq polymerase to each tube
+
*Add 49.5 µL of MM to 4 PCR tubes and add 0.5 µL Taq polymerase to each tube
*J23100-Mlc primer annealing temp- 58.0˚C
*J23100-Mlc primer annealing temp- 58.0˚C
Line 495: Line 511:
*J23100 expected size: 215 bp<br>
*J23100 expected size: 215 bp<br>
-
Did not work
+
*Did not work
==='''May 20: Re-do May 19 PCR of E1010 (fusion) and J23100-mlc'''===  
==='''May 20: Re-do May 19 PCR of E1010 (fusion) and J23100-mlc'''===  
Line 501: Line 517:
Alterations to PCR:  
Alterations to PCR:  
-
*2uL MgCl2
+
*2µL MgCl2
PCR cycle:
PCR cycle:
Line 544: Line 560:
-
*Add 49.5 uL of MM to 4 PCR tubes<br>
+
*Add 49.5 µL of MM to 4 PCR tubes<br>
-
*Add 1uL template DNA<br>
+
*Add 1 µL template DNA<br>
-
*Add 0.5 uL Taq DNA polymerase to each<br>
+
*Add 0.5 µL Taq DNA polymerase to each<br>
Didn’t work (again!)
Didn’t work (again!)
 +
 +
==='''May 21st: PCR of J23100 and E1010 repeat again!'''===  
==='''May 21st: PCR of J23100 and E1010 repeat again!'''===  
Line 585: Line 603:
-
For both J20100 and E1010*:
+
For both J20100 and E1010:
-
*Put 45.9 uL of MM into x PCR tubes<br>
+
*Put 45.9 µL of MM into x PCR tubes<br>
-
*Add 3 uL of template DNA
+
*Add 3 µL of template DNA
*PCR PFU test #4
*PCR PFU test #4
Line 627: Line 645:
PCR Samples:
PCR Samples:
-
[[Image:May_22nd_PCR.JPG|left|417px]]
+
[[Image:May 22nd restriction tubes.JPG|left|px]]
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
Line 650: Line 682:
Ran at 120v for 1 hr
Ran at 120v for 1 hr
-
[[Image:May_22nd_PCR_lanes.JPG|left|px]] [[Image:May_22nd_PCR_gel.JPG|center|246px]]
+
[[Image:May 22nd confirmation gel.JPG|left|px]] [[Image:May_22nd_PCR_gel.JPG|center|246px]]
-
 
+
Line 659: Line 690:
==='''May 23, 2012: Ligation of PCR products into pGEM Vector'''===
==='''May 23, 2012: Ligation of PCR products into pGEM Vector'''===
 +
 +
 +
[[Image:May 23rd PCR tubes E1010 fusion.JPG|left|552px]][[Image:May23.JPG|right|245px]] <br>
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
1) prefix –J23100-mlc-spe cut site (Red tubes) (sample 3 from May 22nd PCR)
1) prefix –J23100-mlc-spe cut site (Red tubes) (sample 3 from May 22nd PCR)
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*2 of each ligated at room temperature for approx. 1hr
*2 of each ligated at room temperature for approx. 1hr
 +
==='''May 23, 2012 Transformation of J23100-Mlc and E1010 fusion'''===
-
 
-
 
-
 
-
 
-
 
-
==='''May 23, 2012 Transformation of J23100-Mlc and E1010 fusion'''===
 
-
[[Image:May23.JPG|right|245px]]
 
For each ligation mix:
For each ligation mix:
Line 697: Line 754:
*Incubate @ 37 ˚C for approximately 16hrs <br>
*Incubate @ 37 ˚C for approximately 16hrs <br>
-
==='''May 23rd continued'''===
 
Make: 1.5% agarose gel  
Make: 1.5% agarose gel  
Line 711: Line 767:
*Add 100bp ladder to two lanes  
*Add 100bp ladder to two lanes  
-
*Middle three lanes ass 5uL of PCR product
+
*Middle three lanes ass 5 µL of PCR product
   
   
*Run at 120V for 1 hour
*Run at 120V for 1 hour
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*45secs in water bath @ 42˚C
*45secs in water bath @ 42˚C
-
*immediately incubated on ice for 5mins
+
*Immediately incubated on ice for 5mins
*Added 400µL of SOC media; resuspended
*Added 400µL of SOC media; resuspended
Line 736: Line 792:
*Incubate @ 37 ˚C for approximately 16hrs
*Incubate @ 37 ˚C for approximately 16hrs
-
 
-
 
-
 
==='''May 24 picking colonies of J20100-mlc and E1010 in DH5ɑ'''===
==='''May 24 picking colonies of J20100-mlc and E1010 in DH5ɑ'''===
Line 744: Line 797:
For each sample:
For each sample:
-
*Pipette 5 uL of ampicillin into 5 mL LB media
+
*Pipette 5 µL of ampicillin into 5 mL LB media
*Pick colony from plate with a pipette tip and eject tip into media
*Pick colony from plate with a pipette tip and eject tip into media
*Shake at 37⁰C for approx. 8h
*Shake at 37⁰C for approx. 8h
-
 
-
 
Line 760: Line 811:
*Resuspend pelleted bacterial cells in 250uL Buffer P1  
*Resuspend pelleted bacterial cells in 250uL Buffer P1  
-
*Add 250uL Buffer P2, mix by inversion
+
*Add 250 µL Buffer P2, mix by inversion
-
*Add 350uL Buffer N3, mix by inversion
+
*Add 350 µL Buffer N3, mix by inversion
*Centrifuge for 10 mins @ 13000 rpm  
*Centrifuge for 10 mins @ 13000 rpm  
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*Centrifuge for 60s at 14000 rpm, discard flow through
*Centrifuge for 60s at 14000 rpm, discard flow through
-
*Add 750uL buffer PE and centrifuge for 60s at 14000 rpm
+
*Add 750 µL buffer PE and centrifuge for 60s at 14000 rpm
*Centrifuge again to get rid of remaining buffer
*Centrifuge again to get rid of remaining buffer
-
*Add 50uL of Buffer EB to spin columns, let stand for I min, centrifuge for 1 min at 14000 rpm
+
*Add 50 µL of Buffer EB to spin columns, let stand for 1 min, centrifuge for 1 min @ 14000 rpm
==='''May 26 gel of Miniprep samples'''===  
==='''May 26 gel of Miniprep samples'''===  
Line 804: Line 855:
*Gel run to check miniprep samples for DNA
*Gel run to check miniprep samples for DNA
* Ran at 120V for 60 minutes
* Ran at 120V for 60 minutes
 +
 +
==='''May 29, 2012: Transformation of E1010 fusion in pGEM in DH5ɑ'''===
==='''May 29, 2012: Transformation of E1010 fusion in pGEM in DH5ɑ'''===
Line 824: Line 877:
*Incubate @ 37 ˚C for approximately 16hrs
*Incubate @ 37 ˚C for approximately 16hrs
 +
 +
==='''May 30 QIA spin Miniprep'''===  
==='''May 30 QIA spin Miniprep'''===  
Line 839: Line 894:
*E1010 p5B2k3
*E1010 p5B2k3
-
*Put 1.5mL of transformed cells in a microcentrifuge tube; centrifuge and repeat until cells from entire tube are in -the microcentrifuge tube.
+
*Put 1.5mL of transformed cells in a microcentrifuge tube; centrifuge and repeat until cells from entire tube are in the microcentrifuge tube.
-
*Resuspend pelleted bacterial cells in 250uL Buffer P1  
+
*Resuspend pelleted bacterial cells in 250µL Buffer P1  
-
*Add 250uL Buffer P2, mix by inversion
+
*Add 250 µL Buffer P2, mix by inversion
-
*Add 350uL Buffer N3, mix by inversion
+
*Add 350 µL Buffer N3, mix by inversion
*Centrifuge for 10 mins @ 13000 rpm  
*Centrifuge for 10 mins @ 13000 rpm  
Line 853: Line 908:
*Centrifuge for 60s at 14000 rpm, discard flow through
*Centrifuge for 60s at 14000 rpm, discard flow through
-
*Add 750uL buffer PE and centrifuge for 60s at 14000 rpm
+
*Add 750 µL buffer PE and centrifuge for 60s at 14000 rpm
*Centrifuge again to get rid of remaining buffer
*Centrifuge again to get rid of remaining buffer
-
*Add 50uL of Buffer EB to spin columns, let stand for I min, centrifuge for 1 min at 14000 rpm
+
*Add 50 µL of Buffer EB to spin columns, let stand for 1 min, centrifuge for 1 min at 14000 rpm
-
 
+
-
 
+
-
 
+
-
 
+
==June==
==June==
Line 881: Line 932:
[[Image:June1.JPG|center|346px]]
[[Image:June1.JPG|center|346px]]
<br>
<br>
 +
 +
===Gel Extraction===
===Gel Extraction===
Line 906: Line 959:
*Transfer 1.5mL of cell culture to microcentrifuge tube; centrifuge at 5000xg for 2 minutes. Repeat three times.
*Transfer 1.5mL of cell culture to microcentrifuge tube; centrifuge at 5000xg for 2 minutes. Repeat three times.
-
*resuspend pelleted cells in 250 µL of Buffer P1
+
*Resuspend pelleted cells in 250 µL of Buffer P1
-
*add 250 µL of buffer P2 and mix by inversion
+
*Add 250 µL of buffer P2 and mix by inversion
-
*add 350 µL of Buffer N1 and mix by inversion
+
*Add 350 µL of Buffer N1 and mix by inversion
-
*centrifuge for 10 minutes at 10000 rpm
+
*Centrifuge for 10 minutes at 10,000 rpm
-
*apply supernatant to the QIAspin column by decanting
+
*Apply supernatant to the QIAspin column by decanting
-
*centrifuge at 13000 prm for 30 seconds
+
*Centrifuge at 13,000 rpm for 30 seconds
-
*wash column by adding 0.75 mL of Buffer PE and centrifuging at 13000 rpm for 30s
+
*Wash column by adding 0.75 mL of Buffer PE and centrifuging at 13,000 rpm for 30 seconds
-
*discard flow through and centriguge for an additional mintue to remove residual buffer
+
*Discard flow through and centriguge for an additional mintue to remove residual buffer
-
*to elute DNA, place column in a microcentrifuge tube, add 50 µL of Buffer EV to centre of column, let stand for 1 minute, centrifuge for 1 minute
+
*To elute DNA, place column in a microcentrifuge tube, add 50 µL of Buffer EV to centre of column, let stand for 1 minute, centrifuge for 1 minute
-
 
+
-
results?
+
==='''June 3, 2012: Confirmaiton of Parts'''===
==='''June 3, 2012: Confirmaiton of Parts'''===
Line 979: Line 1,030:
*B0015 _PSBIAK3
*B0015 _PSBIAK3
 +
 +
==='''June 4, 2012: PCR to Confirm J23100-Mlc in pGEM'''===
==='''June 4, 2012: PCR to Confirm J23100-Mlc in pGEM'''===
Line 1,016: Line 1,069:
*Add 17.8 µL of the Master Mix to four microcentrifuge tubes. <br>
*Add 17.8 µL of the Master Mix to four microcentrifuge tubes. <br>
*Add the template DNA and Pfu polymerase. <br>
*Add the template DNA and Pfu polymerase. <br>
 +
Line 1,024: Line 1,078:
*Incubated on ice for 30mins <br>
*Incubated on ice for 30mins <br>
*Incubated 45secs in water bath @ 42˚C <br>
*Incubated 45secs in water bath @ 42˚C <br>
-
*immediately incubated on ice for 5mins <br>
+
*Immediately incubated on ice for 5mins <br>
*Added 400µL of SOC media; resuspended <br>
*Added 400µL of SOC media; resuspended <br>
*Put in shaker for 1hr @ 37 ˚C <br>
*Put in shaker for 1hr @ 37 ˚C <br>
Line 1,036: Line 1,090:
*Incubated on ice for 30mins <br>
*Incubated on ice for 30mins <br>
*Incubated 45secs in water bath @ 42˚C <br>
*Incubated 45secs in water bath @ 42˚C <br>
-
*immediately incubated on ice for 5mins <br>
+
*Immediately incubated on ice for 5mins <br>
*Added 400µL of SOC media; resuspended <br>
*Added 400µL of SOC media; resuspended <br>
*Put in shaker for 1hr @ 37 ˚C <br>
*Put in shaker for 1hr @ 37 ˚C <br>
*200µL cultured onto plate(4 plates, Amp) <br>
*200µL cultured onto plate(4 plates, Amp) <br>
*Incubate @ 37 ˚C for approximately 16hrs <br>
*Incubate @ 37 ˚C for approximately 16hrs <br>
-
 
-
===Gel Extraction of June 4 Restrictions'''===
 
==='''June 6, 2012: Gel of J23100-Mlc_pGEM Restriction (June 4)'''===
==='''June 6, 2012: Gel of J23100-Mlc_pGEM Restriction (June 4)'''===
Line 1,074: Line 1,126:
*Sample B worked <br>
*Sample B worked <br>
 +
Line 1,091: Line 1,144:
*Put 1.5mL of transformed cells in a microcentrifuge tube; centrifuge and repeat until cells from entire tube are in the microcentrifuge tube. <br>
*Put 1.5mL of transformed cells in a microcentrifuge tube; centrifuge and repeat until cells from entire tube are in the microcentrifuge tube. <br>
*Resuspend pelleted bacterial cells in 250uL Buffer P1 <br>  
*Resuspend pelleted bacterial cells in 250uL Buffer P1 <br>  
-
*Add 250uL Buffer P2, mix by inversion <br>
+
*Add 250µL Buffer P2, mix by inversion <br>
-
*Add 350uL Buffer N3, mix by inversion <br>
+
*Add 350µL Buffer N3, mix by inversion <br>
*Centrifuge for 10 mins @ 13000 rpm <br>
*Centrifuge for 10 mins @ 13000 rpm <br>
*Put supernatant into the QIAprep spin column <br>
*Put supernatant into the QIAprep spin column <br>
-
*Centrifuge for 60s at 14000 rpm, discard flow through <br>
+
*Centrifuge for 60s @ 14000 rpm, discard flow through <br>
-
*Add 750uL buffer PE and centrifuge for 60s at 14000 rpm <br>
+
*Add 750µL buffer PE and centrifuge for 60s @ 14000 rpm <br>
*Centrifuge again to get rid of remaining buffer <br>
*Centrifuge again to get rid of remaining buffer <br>
-
*Add 50uL of Buffer EB to spin columns, let stand for I min, centrifuge for 1 min at 14000 rpm <br>
+
*Add 50µL of Buffer EB to spin columns, let stand for 1 min, centrifuge for 1 min at 14000 rpm <br>
Line 1,145: Line 1,198:
-
[[Image:June6tat.JPG|left|244px]]
+
[[Image:June 6th PCR tubes TAT-E1010.JPG|left|638px]]
-
*TAT-E1010 PCR for insertion into pGEM gel
 
-
*PCR of TAT-E1010 with Taq polymerase
 
-
*Lanes 4, 5, 6 contain the PCR product to be inserted into pGEM
 
-
*1Kb ladder in lane 9
 
Line 1,182: Line 1,231:
-
[[Image:June 6th PCR tubes TAT-E1010.JPG|left|638px]]
 
 +
[[Image:June 6th PCR Gel lanes TAT-E1010.JPG|left|px]][[Image:June6tat.JPG|center|244px]]
 +
*Add 24.25 µL of the Master Mix into 4 microcentrifuge tubes
 +
*Add TAT-E1010 and Taq polymerase
 +
===Size Confirmation Gel of E1010 fusion, TAT-E1010, J23100-Mlc, J04500'''===
 +
[[Image:June 6th size confirmaiton gel lanes.JPG|left|584px]]
Line 1,216: Line 1,269:
-
*Add 24.25 µL of the Master Mix into 4 microcentrifuge tubes
 
-
*Add TAT-E1010 and Taq polymerase
 
-
===Size Confirmation Gel of E1010 fusion, TAT-E1010, J23100-Mlc, J04500'''===
 
-
[[Image:June 6th size confirmaiton gel lanes.JPG|left|584px]]
 
Line 1,242: Line 1,291:
 +
*All TAT-E1010 samples worked
 +
* Sample from lane 5 was ligated with pGEM
 +
===June 7th 2012: Transformation of TAT-E1010_pGEM (x2) and J23100-mlc_pGEM into DH5ɑ===
 +
*Added 2.0 µL of DNA to 20 µL of DH5ɑ.
 +
*Incubated on ice for 30 minutes.
 +
*Placed in water bath for 45 seconds at 42⁰C.
 +
*Immediately incubated in ice for 5 minutes.
 +
*Added 400µL of SOC media, re-suspended.
 +
*Shaked for one hour @ 37⁰C.
 +
*While shaking, 40µL of 0.1M IPTG and 40ul X-Gal was added to each plate
 +
*Added 200µL onto plate.
 +
 +
*Incubated for approximately 16 hours at 37⁰C.
 +
===June 10th 2012: Restriction of J23100-mlc with E and X and J06702 with S and P and Ligation of J23100-mlc with J06702 (pSB1A2) ===
 +
'''Restriction:'''
 +
[[Image:June 10th restriction tubes J06702 PSB1AK3(1).JPG|left|px]]
 +
<br>
 +
[[Image:June_10th_restriction_tubes_J23100-Mlc.JPG|left|px]]
 +
<br>
Line 1,266: Line 1,334:
-
*All TAT-E1010 samples worked
 
-
* Sample from lane 5 was ligated with pGEM
 
-
===June 7th 2012===
 
-
*Transformation
 
-
*2x TAT-E1010(pGem) Amp.
 
-
*1xJ23100-mlc(pGem) Amp.
 
-
*Added 2.0 microlitres, mixed to 20 microlitres of DH5 Alpha.
 
-
*Incubated on ice for 30 minutes.
 
-
*Placed in water bath for 45 seconds at 42 degrees Celsius.
 
-
*Immediately incubated in ice for 5 minutes.
 
-
*Added 400 microlitres of SOC media, re-suspended.
 
-
*Shaked for one hour @ 37 degrees Celsius.
 
-
*While shaking, 40ul of 0.1M IPTG and 40ul X-Gal was added to each plate
 
-
*Added 200 microlitres onto plate.
 
-
 
-
*Incubated for approximately 16 hours at 37 degrees Celsius.
 
-
===June 9th 2012===
 
-
[[Image:June9.JPG|left|225px]]
 
-
*Restriction (X, P) and gel extraction of TAT-E1010
 
-
*Ligation of TAT-E010 with previously gel-extracted J04500 in pSB1AK3 (s,p)
 
Line 1,327: Line 1,375:
 +
*Ran samples on a 2% agarose gel at 120V for 1 hour. <br>
 +
*Only J23100 was seen at the expected size and was extracted from gel according to standard protocol. <br>
 +
*Restrictions were repeated.<br>
 +
[[Image:June_10th_restriction_J06702_repeat.JPG |left|px]]
 +
<br>
-
===June 10th 2012===
+
[[Image:JUne_10th_restriction_J23100_repeat.JPG|left|px]]
-
*Restriction of J23100-mlc with E and X and J06702 with S and P
+
<br>
-
*Ligation of J23100-mlc with J06702 (pSB1A2)
 
-
*Samples A and B were J23100-mlc (gel extracted from June 10th) + J06702
 
-
*Samples C and D were J23100-mlc (normal restrictin) + J06702
 
-
===June 11th 2012===
 
Line 1,359: Line 1,408:
-
*Transformation of J23100-mlc-J06702 samples from June 10th according to standard protocol
+
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
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 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
*Restriced at 37⁰C for 1 hour.<br>
 +
 
 +
 
 +
'''Ligation:'''
 +
 
 +
[[Image:June 10th ligation 1.JPG|left|px]]
 +
 
 +
<br>
 +
 
 +
[[Image:June 10th ligation 2.JPG|left|px]]
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
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 +
 
 +
 
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 +
 
 +
 
 +
 
 +
===June 11th 2012: Transformation of J23100-mlc-J06702 samples from June 10th===
 +
 
 +
For each ligation mix:
 +
 
 +
*Added 2µL of ligation mix to 20.0µL of DH5α
 +
 
 +
*Incubated on ice for 30mins
 +
 
 +
*45secs in water bath @ 42˚C
 +
 
 +
*immediately incubated on ice for 5mins
 +
 
 +
*Added 400µL of SOC media; resuspended
 +
 
 +
*Put in shaker for 1hr @ 37 ˚C
 +
 
 +
*200µL cultured onto plate(4 plates, Amp)
 +
 
 +
*Incubate @ 37 ˚C for approximately 16hr
 +
 
*4 plates (according to June 10th ligation):  
*4 plates (according to June 10th ligation):  
Line 1,392: Line 1,545:
*Incubated for approximately 16 hours at 37 degrees Celsius.
*Incubated for approximately 16 hours at 37 degrees Celsius.
 +
===June 12th, 2012: Picking Cells and Making M9 Media===
-
===June 12th 2012===
 
-
 
-
*Picking cells:
 
*Colonies grew on all 4 plates
*Colonies grew on all 4 plates
*Cells were picked from each plate and put into 5ml LB flasks with 5ul Ampicillin each
*Cells were picked from each plate and put into 5ml LB flasks with 5ul Ampicillin each
 +
 +
*1L M9 media made as per Knight protocol [http://openwetware.org/wiki/Knight:M9_supplemented_media]
 +
 +
Aliquoted as two 10 mL samples of the same glucose concentration. Replacing a portion of the water added with various volumes of 0.693674 mol/L glucose solution as follows:
 +
 +
[[File:M9 pic.jpg]]
 +
 +
==='''June 14th, 2012 PCR of J23100-Mlc-J06702 and J23100-Mlc'''===
 +
 +
[[Image:Screen_Shot_2012-06-16_at_5.58.39_PM.png|left"px]]
 +
 +
 +
-
===June 14th 2012===
 
-
Gel:
 
[[Image:June14.JPG|left|483px]]  
[[Image:June14.JPG|left|483px]]  
-
[[Image:June14gel.JPG|center|199px]]
+
[[Image:June14gel.JPG|center|199px]]<br>
Line 1,417: Line 1,579:
 +
==='''June 15, 2012:Transformation of Plasmid PSB1C3 with J23100-Mlc and K093005 and Insulin Secretion Proof of Princliple'''===
 +
===Transformation:'''===
 +
*Add 20 μL of ligation mix to 20 μL of DH5α
 +
*Incubate on ice for 30 minutes
 +
*30 second s in water bath at 42⁰C
 +
*Immediately incubate in ice for 5 minutes
 +
*Add 200μL of SOC media;resuspend
-
='''Information Sheet'''=
+
*Shake for 1 hour at 37oC for approx. 16 hours
-
='''Protocols'''=
+
===Insulin Secretion Proof of Principle'''===
 +
Picked 4 cultures from -80 freezer stocks: <br>
 +
*2 -J04500 + TAT –E1010 <br>
 +
*2 – J23100 in J61002<br>
 +
Cultures were grown over night <br>
 +
The following morning: <br>
 +
*The 5ml cultures were spun down and re-suspended in one milliliter of fresh media.<br>
 +
*The OD was taken at 600nm and where recorded to determine the amount of the re-suspended  culture was need to inoculate 50ml of media. <br>
-
   
+
[[Image:POP Concentrations of Cell Densities.JPG|left|px]]
 +
<br>
 +
 
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 +
 
 +
 
 +
 
 +
(Note not enough culture was available so 500µL of the 1000µL of the 3rd culture was split between each new culture)
 +
 
 +
The re-suspended cells were added to 50 ml cultures and 50ul of Ampicillin was also added to media. Cultures were placed in the shaker and shaken at 37 ⁰C. <br>
 +
Cultures were measured at 600nm for OD at 1 hour time points from the beginning of shaking.<br>
 +
 
 +
 
 +
[[Image:Culture growth measured by OD at 600nm.JPG|left|px]]
 +
<br>
 +
 
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 +
Culture Samples were spun down and at 8000rpm for 2 minutes and supernatant was taken and ran through a fluorescent spectrophotometer for excitation of RFP at 586nm and expected emission at 607nm.
 +
 
 +
==='''June 16, 2012: PCR of J23100-Mlc-J06702 with Prefix and Suffix (Antisense)'''===
 +
 
 +
 
 +
[[Image:June 16 PCR J23100-Mlc-J06702.JPG|left|px]]
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<html>
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(See [https://2012hs.igem.org/Team:Lethbridge_Canada/Results "Results"].)
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<link rel="stylesheet" href="http://reamsbottom.net/igem/igem_hs.css">
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</html>
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Latest revision as of 03:53, 17 June 2012

Note pic 4.jpg
Home The Team The Project Results Human Practices Notebook Safety



Contents

Notebook

March

Brainstorming Project Ideas

  • Pests
    • Bacterial Pest attractor (engineer bacteria to produce a substance, such as a pheromone or smell, that attracts and kills pests such as insects).
    • Natural pesticides (engineer bacteria to produce a substance that repels or kills pests, such as those that harm crops, that can later be implemented into plants).

  • Water/Environment
    • Desalination of water (engineering bacteria to get rid of salt in salt water in order to make drinkable water from sea water).
    • Getting rid of estrogen mimicking compounds in water (engineering bacteria to degrade them or sequester them)
    • Waste treatment (engineering bacteria that can be integrated into waste water treatment).
    • CFCs (engineer bacteria to produce metabolites that break down chlorofluorocarbons—compounds that contribute to the degradation of the ozone layer).

  • Health
    • Stomach ulcers (creating a medication containing engineered bacteria to specifically target and kill Helicobacter pylori – the organism that causes stomach ulcers).
    • Diabetes (engineered bacteria as Islet cells to produce insulin).
    • Allergies/Immune system (engineer bacteria to produce antihistamines or alter epitopes).
    • Bone density (engineer bacteria to produce and secrete calcium and other compounds to help heal broken bones or to prevent osteoporosis).

  • Process Improvement
    • Oil fractionation catalyst (engineering bacteria to improve the separation of crude oil into valuable fraction and waste fraction).
    • Nitrate fixation (engineer bacteria to improve nitrogen fixation so not as much fertilizer is needed).

  • Kill Switches
    • Engineering bacteria to undergo induced or programmed cell death in order to control the organism).

The chosen project

  • After dividing in to a few groups based on which project was preferred, each group made a presentation on the proposed project. In the end, we decided to go with the glucose sensing project.

Project outline

Possible things to consider:

  • Glucose and insulin
    • Detection of blood glucose levels (glucose-specific promoter?)
    • Insulin production release (measure/regulation of production?)

  • Immune response
    • Possibly use "bactoblood" strain from Berkeley 2007

  • Kill switch
  • Delivery system
    • Intravenous
    • NASA Biocapsule

April

Sub-groups

We divided the team into sub-groups. Each group assumed responsibility for a different aspect of the project.

Wiki and Mascot Design

This group is responsible to obtaining and uploading all necessary information to the wiki. This includes team pictures and biographies, experiments and results, and the projects of the other sub-groups. This will be a priority over mascot design.
Leader: Erin Kelly
1. Erin Kwan
2. Alycia Amatto
3. Cassandra Logue
4. Teddi Reynolds
5. Dax Law
6. Riley Martens
7. Elaine Bird
8. Brooke Heatherington
9. Corbin Chenger
10. Devan Carrier


Biosafety

This group must document the safety protocols that the Lethbridge team practices in the lab.
Leader: Marissa Guzzi
1. Branden Black
2. Iain Sander
3. Carissa Kirk
4. Brianna Carrels
5. Amanda
6. Alli Herauf


Human Practices

This group is responsible for letting the public know what our team is and what we are doing. This can be done creatively. The point is to raise awareness for iGEM, synthetic biology, and diabetes.
Leader: Chris Isaac
1. Shammamah Hossain
2. Dawson Meyer
3. Yoyo Yao
4. Jared Sparkes
5. Wesley Mosimann
6. Trisha Bouma
7. Janelle Veenendaal
8. Orion Sehn

WHMIS tests and Safety Training

We are required to have safety training in order to work in the lab, and so we all wrote our WHMIS tests and did our Hazards Assesment and lab orientation!

May

May 8, 2012: Tansformation of Parts from Kit Plate and Growing Cells from Glycerol Stocks

Transformation of parts from kit plate:

  • B0034 in pSB1A2
  • K093005 in pSB1A2
  • J23100 in J61002 (Amp)
  • E1010 in pSB2K3


2µL of DNA was added to 20µL DH5ɑ cells

  • Incubated on ice for 30 mins
  • Cells were then heat shocked for 45sec in water bath at 42 degrees Celsius
  • Immediately after, cells were incubated on ice for 5 mins
  • 400µL SOC media was added and cells were resuspended
  • Cells were then placed in shaker for 1h @ 37 degrees Celsius
  • 200µL of this culture was plated on AMP plates
  • Plates were incubated at 37 degrees for appx. 16hrs.


Overnight cultures were also made of:

  • pMA-T K33109 (HST-1)
  • pSB1AK3 B0014

May 9, 2012: Miniprep and Restriction of Parts

Overnight cultures from May 8th were miniprepped and restricted in order to determine sizes.


May 9th restriction tubes.JPG













Master mix was prepared by mixing the ingredients together in order, as shown above. 20µL worth of master mix was combined with 5µL of plasmid DNA:

  • pMA-T BBa_K331009 (Heat-stable toxin signal sequence)
  • pSB1AK3 BBa_B0014 (Double terminator)

Restrictions were incubated at 37˚C for 30mins then stored at -20˚C.


Colonies were also picked. Overnight cultures were made of :

  • E1010 pSB2K3
  • J23100 J61002 (Amp)
  • K93005 pSB1A2
  • B0034 pSB1A2


May 10, 2012: Miniprep and Restriction of Parts

Overnight cultures from May 9th were miniprepped and restricted in order to determine sizes

May 10th restriction tubes.JPG













Master mix was prepared by mixing the ingredients together in order, as shown above. 20µL worth of master mix was combined with 5µL of plasmid DNA (1-7).

  • J23100 (J61002)
  • J23100 (J61002)
  • B0034 (pSB1A3)
  • B0034 (pSB1A3)
  • E1010 (pSB2K3)
  • E1010 (pSB2k3)
  • K093008 (pSB1A2)

Restrictions were incubated at 37˚C for 30min then stored in -20˚C.


May 11th: Electrophoresis of restricted parts from May 10th


May 11th PCR tubes.JPG














Purpose: confirm existence of the following parts:

  • J23100 (J61002)
  • B0034 (pSB1A3)
  • E1010 (pSB2K3)
  • K093008 (pSB1A2)


  • We also grew up J04500 in pSB1AK3 for assembly


PCR of RFP (E1010) into fusion standard

(adding extra bp to get into fusion standard) Add 17.8 µL to 8 tubes with:
  • Pfu polymerase 0.2 µL
  • Template DNA 2 µL
  • Put tubes on gradient function program Pfu test with various annealing temperatures:
Annealing gradient (degrees celsius):
  • 49.2
  • 51.6
  • 53.0
  • 54.5
  • 55.9
  • 57.3
  • 58.8
  • 61.2
  • only 54.5 worked

May 12th: Assembly of promoter & RBS (J04500) with signal sequence (K331009) and RPF (E1010 fusion) with double terminator (B0015)

Method: Restriction, gel extraction, and ligation

Restriction:

  • J04500
  • K331009
  • E1010 (fusion)
  • B0015


Gel extraction:

We made a 1% TAE agarose gel and ran restrictions for 1 hour @ 120V

We then gel extracted the DNA

Ligation:

  • Ligated together J04500 & K331009
  • Ligated together E1010 fusion & B0015


May 13th Transformation of ligated products: J04500 + K331009 and E1010 (fusion) + B0015

Transformation:

2µL of each ligation mix was added to 20µL DH5ɑ cells

  • Incubated on ice for 30 mins
  • Cells were then heat shocked for 45sec in water bath at 42 degrees Celsius
  • Immediately after, cells were incubated on ice for 5 mins
  • 400µL SOC media was added and cells were resuspended
  • Cells were then placed in the shaker for 1h @ 37 degrees
  • 200µL of this culture was plated on AMP plates
  • Plates were incubated at 37 degrees for appx. 16hrs.

May 14th: Picking Cells from May 13th transformation

  • Cells grew on 2 plates, 2 colonies were picked
  • Picked cells were put into 5ml LB media with kanamycin
  • Cells did not grow


May 19th: PCR of E1010 and J23100 (Mlc primers)

E1010:

May 19th PCR E1010.JPG
















  • Add 49.5µL to 4 PCR tubes and add 0.5µL Taq polymerase and 1µL template DNA to each
  • E1010 (fusion) primer
  • The annealing temp was 55.5˚C



J23100:

May 19th PCR J23100-Mlc.JPG
















  • Add 49.5 µL of MM to 4 PCR tubes and add 0.5 µL Taq polymerase to each tube
  • J23100-Mlc primer annealing temp- 58.0˚C


Ran 1.5% agarose TAE gel at 90V for 45 mins

May 19th gel extraction.JPG























  • E1010 expected size: 700 bp
  • J23100 expected size: 215 bp
  • Did not work

May 20: Re-do May 19 PCR of E1010 (fusion) and J23100-mlc

Alterations to PCR:

  • 2µL MgCl2

PCR cycle:

  • First Denaturation: 3 mins @ 94˚
  • Denaturation: 15 sec @94˚
  • Annealing: 30sec E1010: 54.5˚ J23100: 55.0˚
  • Elongation: I min 30 sec @ 72˚
  • Final elongation: 7 mins @72˚


May 20th PCR E1010.JPG
















  • Add 49.5 µL of MM to 4 PCR tubes
  • Add 1 µL template DNA
  • Add 0.5 µL Taq DNA polymerase to each


Didn’t work (again!)


May 21st: PCR of J23100 and E1010 repeat again!

May 21st PCR E1010 and J23100.JPG
















For both J20100 and E1010:

  • Put 45.9 µL of MM into x PCR tubes
  • Add 3 µL of template DNA
  • PCR PFU test #4
  • Ran on 1.5% agarose gel at 120V for 1hour

PCR protocol:

  • First Denaturation: 2 mins @ 95˚
  • Denaturation: 30 sec @95˚
  • Annealing: 30sec E1010: 54.5˚ J23100: 55.0˚
  • Elongation: I min 30 sec @ 72˚
  • First elongation: 7 mins @72˚
  • changed to 30 cycles

May 22, 2012: PCR of J23100-Mlc

PCR Parameters:

Initial denaturation 95˚C 3mins 30 cycles of:

*denaturation 95˚C 30s

*annealing (various) 30s

*elongation 72˚C 1min


Final elongation 72˚C 7mins


PCR Samples:

px
















PCR product expected size: 214 bps

1.5% agarose gel

Ran at 120v for 1 hr

px
May 22nd PCR gel.JPG




May 23, 2012: Ligation of PCR products into pGEM Vector

May 23rd PCR tubes E1010 fusion.JPG
May23.JPG
















1) prefix –J23100-mlc-spe cut site (Red tubes) (sample 3 from May 22nd PCR)

2) Prefix-(fusion)-E1010-suffix (Black tubes) (sample 1 from May 23rd PCR)

Contents:

  • 2x Rapid Ligation Buffer 5µL
  • pGEM-Tor pGem-T Easy Vector 1µL
  • PCR Product 3 µL
  • T4 DNA Ligase 1µL
  • MiliQ H2O 1 µL
  • Ligate at room temp for 1h 15 mins
  • One of each sample ligated overnight @ 4˚c
  • 2 of each ligated at room temperature for approx. 1hr

May 23, 2012 Transformation of J23100-Mlc and E1010 fusion

For each ligation mix:

  • Added 2µL of ligation mix ( E1010 pcr + pGEM; J23100- mlc + pGEM) to 20.0µL of DH5α)
  • Incubated on ice for 30mins
  • 45secs in water bath @ 42˚C
  • immediately incubated on ice for 5mins
  • Added 400µL of SOC media; resuspended
  • Put in shaker for 1hr @ 37 ˚C
  • 200µL cultured onto plate(4 plates, Amp)
  • Incubate @ 37 ˚C for approximately 16hrs


Make: 1.5% agarose gel

-0.45 agarose M

-30 mL TAE (1x)


  • Run PCR samples 1, 3, & 4 from PCR (E1010  RFP)
  • Add 100bp ladder to two lanes
  • Middle three lanes ass 5 µL of PCR product
  • Run at 120V for 1 hour

May 24, 2012: Transformation of PCR Products in pGEM Vectors in DH5ɑ

Sample 3 from PCR product ligation

For each ligation mix:

  • Added 2µL of ligation mix ( E1010 pcr + pGEM; J23100- mlc + pGEM) to 20.0µL of DH5α
  • Incubated on ice for 30mins
  • 45secs in water bath @ 42˚C
  • Immediately incubated on ice for 5mins
  • Added 400µL of SOC media; resuspended
  • Put in shaker for 1hr @ 37 ˚C
  • 200µL cultured onto plate(4 plates, Amp)
  • Incubate @ 37 ˚C for approximately 16hrs

May 24 picking colonies of J20100-mlc and E1010 in DH5ɑ

For each sample:

  • Pipette 5 µL of ampicillin into 5 mL LB media
  • Pick colony from plate with a pipette tip and eject tip into media
  • Shake at 37⁰C for approx. 8h


May 25, 2012: QIA Spin Miniprep of J23100-mlc (A: colony 1), J23100-mlc (B: colony 2)

  • Put 1.5mL of transformed cells in a microcentrifuge tube; centrifuge and repeat until cells from entire tube are in the microcentrifuge tube.
  • Resuspend pelleted bacterial cells in 250uL Buffer P1
  • Add 250 µL Buffer P2, mix by inversion
  • Add 350 µL Buffer N3, mix by inversion
  • Centrifuge for 10 mins @ 13000 rpm
  • Put supernatant into the QIAprep spin column
  • Centrifuge for 60s at 14000 rpm, discard flow through
  • Add 750 µL buffer PE and centrifuge for 60s at 14000 rpm
  • Centrifuge again to get rid of remaining buffer
  • Add 50 µL of Buffer EB to spin columns, let stand for 1 min, centrifuge for 1 min @ 14000 rpm

May 26 gel of Miniprep samples

May 26th miniprep gel.JPG













  • Gel run to check miniprep samples for DNA
  • Ran at 120V for 60 minutes


May 29, 2012: Transformation of E1010 fusion in pGEM in DH5ɑ

For each ligation mix:

  • Added 2µL of ligation mix ( E1010 pcr + pGEM; J23100- mlc + pGEM) to 20.0µL of DH5α
  • Incubated on ice for 30mins
  • 45secs in water bath @ 42˚C
  • immediately incubated on ice for 5mins
  • Added 400µL of SOC media; resuspended
  • Put in shaker for 1hr @ 37 ˚C
  • 200µL cultured onto plate(4 plates, Amp)
  • Incubate @ 37 ˚C for approximately 16hrs


May 30 QIA spin Miniprep

DNA:

  • E1010 psB2k3
  • E1010 fusion pGEM
  • J20100-mlc pGEM
  • CK331009 psEk3
  • E1010 p5B2k3
  • Put 1.5mL of transformed cells in a microcentrifuge tube; centrifuge and repeat until cells from entire tube are in the microcentrifuge tube.
  • Resuspend pelleted bacterial cells in 250µL Buffer P1
  • Add 250 µL Buffer P2, mix by inversion
  • Add 350 µL Buffer N3, mix by inversion
  • Centrifuge for 10 mins @ 13000 rpm
  • Put supernatant into the QIAprep spin column
  • Centrifuge for 60s at 14000 rpm, discard flow through
  • Add 750 µL buffer PE and centrifuge for 60s at 14000 rpm
  • Centrifuge again to get rid of remaining buffer
  • Add 50 µL of Buffer EB to spin columns, let stand for 1 min, centrifuge for 1 min at 14000 rpm

June

June 1, 2012: Restriction of J23100-Mlc, K093005, B0014 (samples 1 and 2), K331009_PSB1C3, K331009, E1010 Fusion (samples 1 and 2), J04500


June 1st restriction J23100-Mlc.JPG
June 1st restriction K093005.JPG


June 1st restriction B0014.JPG
June 1st restriction B0014 (2).JPG


June 1st restriction K331009 PSB1C3.JPG
June 1st restriction K331009.JPG


June 1st restriction E1010 Fusion.JPG
June 1st restriction E1010 Fusion (2).JPG


JUne 1st restriction J04500.JPG


June1.JPG



Gel Extraction

June1ge.JPG






  • Only J04500 (lane 6) and K331009 (lane 9) were the expected size
  • Restrictions were incubated at 37˚C for 30min then stored in -20˚C


June 2, 2012: Miniprep of June 1 Cultures

  • Transfer 1.5mL of cell culture to microcentrifuge tube; centrifuge at 5000xg for 2 minutes. Repeat three times.
  • Resuspend pelleted cells in 250 µL of Buffer P1
  • Add 250 µL of buffer P2 and mix by inversion
  • Add 350 µL of Buffer N1 and mix by inversion
  • Centrifuge for 10 minutes at 10,000 rpm
  • Apply supernatant to the QIAspin column by decanting
  • Centrifuge at 13,000 rpm for 30 seconds
  • Wash column by adding 0.75 mL of Buffer PE and centrifuging at 13,000 rpm for 30 seconds
  • Discard flow through and centriguge for an additional mintue to remove residual buffer
  • To elute DNA, place column in a microcentrifuge tube, add 50 µL of Buffer EV to centre of column, let stand for 1 minute, centrifuge for 1 minute

June 3, 2012: Confirmaiton of Parts

JUne 3rd size confirmation gel lanes (2).JPG



















What worked:

  • B0015_PSBIAK3
  • K331009_PSBIC3
  • J04500_PSBIAK3
  • B0015_PSBIAK3
  • J04500_PSBIAK3
  • B0015 _PSBIAK3


June 4, 2012: PCR to Confirm J23100-Mlc in pGEM

June 4th PCR J23100-Mlc pGEM.JPG
















  • Add 17.8 µL of the Master Mix to four microcentrifuge tubes.
  • Add the template DNA and Pfu polymerase.


Transformation of TAT-E1010_pGEM (x2) and J23100-Mlc_pGEM (x1) into DH5α

For each ligation mix:

  • Added 2µL of ligation mix ( E1010 pcr + pGEM; J23100- mlc + pGEM) to 20.0µL of DH5α
  • Incubated on ice for 30mins
  • Incubated 45secs in water bath @ 42˚C
  • Immediately incubated on ice for 5mins
  • Added 400µL of SOC media; resuspended
  • Put in shaker for 1hr @ 37 ˚C
  • 200µL cultured onto plate(4 plates, Amp)
  • Incubate @ 37 ˚C for approximately 16hrs

June 5, 2012: Transformation of J06702 (mCherry RFP) into DH5α

  • Add 10µL of MilliQ H2O into Kit Plate (Spring 2011, Kit Plate #2, well E8)
  • Added 2µL of DNA to 20.0µL of DH5α
  • Incubated on ice for 30mins
  • Incubated 45secs in water bath @ 42˚C
  • Immediately incubated on ice for 5mins
  • Added 400µL of SOC media; resuspended
  • Put in shaker for 1hr @ 37 ˚C
  • 200µL cultured onto plate(4 plates, Amp)
  • Incubate @ 37 ˚C for approximately 16hrs

June 6, 2012: Gel of J23100-Mlc_pGEM Restriction (June 4)

June 6th PCR Gel lanes J23100-Mlc.JPG














  • Sample B worked


Miniprep of J06702

June6.JPG


  • Gel confirming J06702 miniprep
  • Conformation of part BBa_J06702
  • Miniprep sample cut with EcoR1 and ran on a 1% agarose gel at 120V for 50 mins
  • Lanes 4, 5, and 6 contain restricted J06702 in pGEM


  • Put 1.5mL of transformed cells in a microcentrifuge tube; centrifuge and repeat until cells from entire tube are in the microcentrifuge tube.
  • Resuspend pelleted bacterial cells in 250uL Buffer P1
  • Add 250µL Buffer P2, mix by inversion
  • Add 350µL Buffer N3, mix by inversion
  • Centrifuge for 10 mins @ 13000 rpm
  • Put supernatant into the QIAprep spin column
  • Centrifuge for 60s @ 14000 rpm, discard flow through
  • Add 750µL buffer PE and centrifuge for 60s @ 14000 rpm
  • Centrifuge again to get rid of remaining buffer
  • Add 50µL of Buffer EB to spin columns, let stand for 1 min, centrifuge for 1 min at 14000 rpm


  • 3 samples were made and ran on a gel, all samples appeared to be the expected size

PCR of E1010 Fusion (to confirm part) and TAT-E1010 (for insertion into pGEM)

June 6th PCR tubes E1010 fusion.JPG

















  • Add 18.8 µL of the Master Mix into 4 microcentrifuge tubes
  • Add E1010 Fusion and Pfu polymerase



June 6th PCR tubes TAT-E1010.JPG



















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June6tat.JPG



  • Add 24.25 µL of the Master Mix into 4 microcentrifuge tubes
  • Add TAT-E1010 and Taq polymerase


Size Confirmation Gel of E1010 fusion, TAT-E1010, J23100-Mlc, J04500

June 6th size confirmaiton gel lanes.JPG






















  • All TAT-E1010 samples worked
  • Sample from lane 5 was ligated with pGEM

June 7th 2012: Transformation of TAT-E1010_pGEM (x2) and J23100-mlc_pGEM into DH5ɑ

  • Added 2.0 µL of DNA to 20 µL of DH5ɑ.
  • Incubated on ice for 30 minutes.
  • Placed in water bath for 45 seconds at 42⁰C.
  • Immediately incubated in ice for 5 minutes.
  • Added 400µL of SOC media, re-suspended.
  • Shaked for one hour @ 37⁰C.
  • While shaking, 40µL of 0.1M IPTG and 40ul X-Gal was added to each plate
  • Added 200µL onto plate.
  • Incubated for approximately 16 hours at 37⁰C.

June 10th 2012: Restriction of J23100-mlc with E and X and J06702 with S and P and Ligation of J23100-mlc with J06702 (pSB1A2)

Restriction:

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  • Ran samples on a 2% agarose gel at 120V for 1 hour.
  • Only J23100 was seen at the expected size and was extracted from gel according to standard protocol.
  • Restrictions were repeated.


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  • Restriced at 37⁰C for 1 hour.


Ligation:

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June 11th 2012: Transformation of J23100-mlc-J06702 samples from June 10th

For each ligation mix:

  • Added 2µL of ligation mix to 20.0µL of DH5α
  • Incubated on ice for 30mins
  • 45secs in water bath @ 42˚C
  • immediately incubated on ice for 5mins
  • Added 400µL of SOC media; resuspended
  • Put in shaker for 1hr @ 37 ˚C
  • 200µL cultured onto plate(4 plates, Amp)
  • Incubate @ 37 ˚C for approximately 16hr


  • 4 plates (according to June 10th ligation):
  • A
  • B
  • C
  • D


  • 2x J23100-m/c (gel extraction) Amp.
  • 2x J06702 Amp.
  • Added 2.0 microlitres, mixed to 20 microlitres of DH5 Alpha.
  • Incubated on ice for 30 minutes.
  • Placed in water bath for 45 seconds at 42 degrees Celsius.
  • Immediately incubated in ice for 5 minutes.
  • Added 400 microlitres of SOC media, re-suspended.
  • Shook for one hour @ 37 degrees Celsius.
  • Added 200 microlitres onto plate.
  • Incubated for approximately 16 hours at 37 degrees Celsius.

June 12th, 2012: Picking Cells and Making M9 Media

  • Colonies grew on all 4 plates
  • Cells were picked from each plate and put into 5ml LB flasks with 5ul Ampicillin each
  • 1L M9 media made as per Knight protocol [http://openwetware.org/wiki/Knight:M9_supplemented_media]

Aliquoted as two 10 mL samples of the same glucose concentration. Replacing a portion of the water added with various volumes of 0.693674 mol/L glucose solution as follows:

M9 pic.jpg

June 14th, 2012 PCR of J23100-Mlc-J06702 and J23100-Mlc

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June14.JPG
June14gel.JPG





June 15, 2012:Transformation of Plasmid PSB1C3 with J23100-Mlc and K093005 and Insulin Secretion Proof of Princliple

Transformation:

  • Add 20 μL of ligation mix to 20 μL of DH5α
  • Incubate on ice for 30 minutes
  • 30 second s in water bath at 42⁰C
  • Immediately incubate in ice for 5 minutes
  • Add 200μL of SOC media;resuspend
  • Shake for 1 hour at 37oC for approx. 16 hours

Insulin Secretion Proof of Principle

Picked 4 cultures from -80 freezer stocks:

  • 2 -J04500 + TAT –E1010
  • 2 – J23100 in J61002

Cultures were grown over night
The following morning:

  • The 5ml cultures were spun down and re-suspended in one milliliter of fresh media.
  • The OD was taken at 600nm and where recorded to determine the amount of the re-suspended culture was need to inoculate 50ml of media.


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(Note not enough culture was available so 500µL of the 1000µL of the 3rd culture was split between each new culture)

The re-suspended cells were added to 50 ml cultures and 50ul of Ampicillin was also added to media. Cultures were placed in the shaker and shaken at 37 ⁰C.
Cultures were measured at 600nm for OD at 1 hour time points from the beginning of shaking.


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Culture Samples were spun down and at 8000rpm for 2 minutes and supernatant was taken and ran through a fluorescent spectrophotometer for excitation of RFP at 586nm and expected emission at 607nm.

June 16, 2012: PCR of J23100-Mlc-J06702 with Prefix and Suffix (Antisense)

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(See "Results".)