Team:Michigan/Oil Sands October
From 2010.igem.org
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- | [[Team:Michigan/Oil_Sands | Back to Oil Sands Main]] | + | [[Team:Michigan/Oil_Sands | Back to Oil Sands Main Notebook]] |
- | + | ||
==10/1/2010== | ==10/1/2010== | ||
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Moved broth cultures 37°C to 4°C -- 1:30pm (19-hr culture) | Moved broth cultures 37°C to 4°C -- 1:30pm (19-hr culture) | ||
+ | |||
Prepared biofilm assay plate for all pH9 tests - following [[Media:Static+biofilm+quantification.pdf|Alex's biofilm assay protocol]] | Prepared biofilm assay plate for all pH9 tests - following [[Media:Static+biofilm+quantification.pdf|Alex's biofilm assay protocol]] | ||
*plate template: | *plate template: | ||
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---- | ---- | ||
Saved data for curves from ph7 plate | Saved data for curves from ph7 plate | ||
+ | |||
Read pH7 plate ODs | Read pH7 plate ODs | ||
+ | |||
Performed biofilm assay on ph7 plate | Performed biofilm assay on ph7 plate | ||
+ | |||
Started 24-hr kinetic reading for pH9 plate -- ~midnight | Started 24-hr kinetic reading for pH9 plate -- ~midnight | ||
+ | |||
Discarded pH7 plate | Discarded pH7 plate | ||
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*Calculated amount from digests to add to ligation for 1:1 and 3:1 insert:vector ratios | *Calculated amount from digests to add to ligation for 1:1 and 3:1 insert:vector ratios | ||
**Amount of DNA in digests: | **Amount of DNA in digests: | ||
- | ***Flu PCR- 29.8 ng/uL x 17 uL= 506.6 ng / 50 uL = 10.132 ng/uL | + | ***Flu PCR - 29.8 ng/uL x 17 uL = 506.6 ng / 50 uL = 10.132 ng/uL |
- | ***pSB1AT3- 240.5 ng/uL x 5 uL= 1202.5 ng / 50 uL = 24.05 ng/uL | + | ***pSB1AT3 - 240.5 ng/uL x 5 uL = 1202.5 ng / 50 uL = 24.05 ng/uL |
**Used an online calculator to find moles: | **Used an online calculator to find moles: | ||
- | ***Flu PCR- 230.81 fmol/50 uL | + | ***Flu PCR - 230.81 fmol/50 uL |
- | ***pSB1AT3- 537.68 fmol/50 uL | + | ***pSB1AT3 - 537.68 fmol/50 uL |
***This gives a molar ratio of 0.4293 | ***This gives a molar ratio of 0.4293 | ||
- | 1:1 Ligation Mix | + | 1:1 Ligation Mix: |
- | *4 uL Flu PCR x 10.132 ng/uL= 40.528 ng | + | *4 uL Flu PCR x 10.132 ng/uL = 40.528 ng |
- | *1.7 uL pSB1AT3 x 24.05 ng/uL= 40.885 ng | + | *1.7 uL pSB1AT3 x 24.05 ng/uL = 40.885 ng |
*2 uL T4 Buffer | *2 uL T4 Buffer | ||
*1 uL T4 Ligase | *1 uL T4 Ligase | ||
*11.3 uL Ultra pure H2O | *11.3 uL Ultra pure H2O | ||
- | 3:1 Ligation Mix | + | 3:1 Ligation Mix: |
- | *2 uL Flu PCR x 10.132 ng/uL= 20.264 ng | + | *2 uL Flu PCR x 10.132 ng/uL = 20.264 ng |
- | *2.57 uL pSB1AT3 x 24.05 ng/uL= 61.809 ng | + | *2.57 uL pSB1AT3 x 24.05 ng/uL = 61.809 ng |
*2 uL T4 Buffer | *2 uL T4 Buffer | ||
*1 uL T4 Ligase | *1 uL T4 Ligase | ||
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Performed transformation according to [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC145660/pdf/240536.pdf Pope & Kent (1996)]. | Performed transformation according to [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC145660/pdf/240536.pdf Pope & Kent (1996)]. | ||
- | Transformed Parts | + | Transformed Parts: |
*Flu/pSB1AT3 1:1 ligation | *Flu/pSB1AT3 1:1 ligation | ||
*Flu/pSB1AT3 1:3 ligation | *Flu/pSB1AT3 1:3 ligation | ||
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*Cell only negative control | *Cell only negative control | ||
- | Procedure | + | Procedure: |
*2 uL of DNA was pipetted into PCR tube on ice and transferred to Lin lab | *2 uL of DNA was pipetted into PCR tube on ice and transferred to Lin lab | ||
*Comp cells were thawed on ice and 50 uL was pipetted into each tube with a chilled pipette on ice | *Comp cells were thawed on ice and 50 uL was pipetted into each tube with a chilled pipette on ice | ||
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**Kevin put plates in incubator several hours before hand | **Kevin put plates in incubator several hours before hand | ||
- | It should be noted that two 200 uL aliquots of cells had to be thawed, and one was labeled "Thawed" and returned to the - | + | It should be noted that two 200 uL aliquots of cells had to be thawed, and one was labeled "Thawed" and returned to the -80ºC. Thawing should be avoided if possible. |
- | + | Two 5 mL LD1/LD2 co-cultures in M9+Sigma and Acros NAs were made: pH 7 and pH 9. Also made 2 mL pure cultures at pH 7. All culture tubes were stored in the 30ºC. These cultures were made to assay growth, make new stock plates, and determine if colony morphology can be used to distinguish between species in a co-culture. | |
== 10/7/2010 == | == 10/7/2010 == | ||
''Marcus'' | ''Marcus'' | ||
- | Ben moved transformation plates to | + | Ben moved transformation plates to 4ºC yesterday, control plates were blank, and all plates except for J61100 appeared to have colonies. The Flu/pSB1AT3 ligation plates had very small colonies, so they were returned to the incubator with the J61100 plate. Also, it could be seen that many colonies on the 1:3 ligation plate appeared red, indicating that the vector had preferentially reformed. This could be because of the ratio, but dephosphorylation treatment should be used in the future for ligations. |
- | + | OD600s were taken of the Pseudomonas cultures (46 hours): | |
- | *Putida pH 7- 0.157 | + | *Putida pH 7 - 0.157 |
- | *Fluorescens pH 7- 0.129 | + | *Fluorescens pH 7 - 0.129 |
- | *Co-culture pH 7- 0.188 | + | *Co-culture pH 7 - 0.188 |
- | *Co-culture pH 9- 0.232 | + | *Co-culture pH 9 - 0.232 |
Surprisingly, they seem to grow better at pH 9, at least in co-culture. As expected, the co-culture did better than pure cultures. This lends some support towards Alex's biofilm assay results, however, it should still be repeated. Also, plated a serial dilution of the pH 9 co-culture, at 10<sup>-5</sup>, 10<sup>-6</sup>, and 10<sup>-7</sup> dilutions to determine the conversion factor between OD600 and cell count. Made streak plates of all other cultures, pure cultures for stock, and pH 7 co-culture to determine if colonies could be distinguished. All cultures and plates were returned to the 30<sup>o</sup>C incubator, without shaking. | Surprisingly, they seem to grow better at pH 9, at least in co-culture. As expected, the co-culture did better than pure cultures. This lends some support towards Alex's biofilm assay results, however, it should still be repeated. Also, plated a serial dilution of the pH 9 co-culture, at 10<sup>-5</sup>, 10<sup>-6</sup>, and 10<sup>-7</sup> dilutions to determine the conversion factor between OD600 and cell count. Made streak plates of all other cultures, pure cultures for stock, and pH 7 co-culture to determine if colonies could be distinguished. All cultures and plates were returned to the 30<sup>o</sup>C incubator, without shaking. | ||
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PCR Screening: | PCR Screening: | ||
- | 8 colonies were picked from each transformation plate except for VP130 | + | 8 colonies were picked from each transformation plate except for VP130 and suspended in 50 uL of water on a microplate.: |
*Plate Layout | *Plate Layout | ||
- | **Row 1- Flu 1:1 | + | **Row 1 - Flu 1:1 |
- | **Row 2- Flu 1:3 | + | **Row 2 - Flu 1:3 |
- | **Row 3- OmpA | + | **Row 3 - OmpA |
- | **Row 4- J61100 | + | **Row 4 - J61100 |
- | The first two rows (Flu 1:1 and 1:3) were PCR'd by adding 1 uL of each sample to a PCR tube with a drop of mineral oil in them. A master mix was made | + | The first two rows (Flu 1:1 and 1:3) were PCR'd by adding 1 uL of each sample to a PCR tube with a drop of mineral oil in them. A master mix was made and 50 uL was added to each tube (should have been 49 uL). |
- | Master Mix (18x) | + | Master Mix (18x): |
*180 uL 5x Phusion Buffer | *180 uL 5x Phusion Buffer | ||
*18 uL 10 mM dNTPs | *18 uL 10 mM dNTPs | ||
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*585 uL Ultra pure H2O | *585 uL Ultra pure H2O | ||
- | PCR Program | + | PCR Program: |
- | *1. | + | *1. 95ºC for 10 min |
- | *2. | + | *2. 95ºC for 30 sec |
- | *3. | + | *3. 62ºC for 30 sec |
- | *4. | + | *4. 72ºC for 2 min |
*5. Return to step two 34 times | *5. Return to step two 34 times | ||
- | *6. | + | *6. 72ºC for 10 min |
- | *7. | + | *7. 4ºC store |
This PCR protocol was constructed from the following sources: | This PCR protocol was constructed from the following sources: | ||
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*[http://www.finnzymes.com/pdf/f530sl_phusion_datasheet_2_2_low.pdf Finnzymes Phusion Manual] | *[http://www.finnzymes.com/pdf/f530sl_phusion_datasheet_2_2_low.pdf Finnzymes Phusion Manual] | ||
- | Also- loosened the lids on Pseudomonas liquid cultures to provide air exchange, and returned yesterday's Pseduomonas plates to | + | Also- loosened the lids on Pseudomonas liquid cultures to provide air exchange, and returned yesterday's Pseduomonas plates to 30ºC incubator, without shaking. |
== 10/11/2010 == | == 10/11/2010 == | ||
''Marcus'' | ''Marcus'' | ||
- | Ran a gel of PCR products from screening. Ran gel according to standard [[Media:Protocol_for_Running_a_Gel.pdf|Gel Electrophoresis]] protocol, for 45 minutes at | + | Ran a gel of PCR products from screening. Ran gel according to standard [[Media:Protocol_for_Running_a_Gel.pdf|Gel Electrophoresis]] protocol, for 45 minutes at 86 V. |
[[Image:Flu Colony Screening Annotated.png|250px]] | [[Image:Flu Colony Screening Annotated.png|250px]] | ||
- | Samples 7 and 8 from the 1:3 ligation appeared to be promising (expected band size= 3655 bp), so they were plated and stored in the | + | Samples 7 and 8 from the 1:3 ligation appeared to be promising (expected band size= 3655 bp), so they were plated and stored in the 37ºC incubator overnight. |
== 10/13/2010 == | == 10/13/2010 == | ||
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''Marcus'' | ''Marcus'' | ||
- | Overnights made were only 2 mL, and should've been 5 mL. So 3 mL of fresh LB (with antibiotics) was added to 50 mL tubes, and the cultures were added to the fresh LB at 1 pm. Minipreps were made according to the [[Media:MODIFIED_Miniprep_Protocol.pdf|MODIFIED Miniprep]] at room temperature and stored in the | + | Overnights made were only 2 mL, and should've been 5 mL. So 3 mL of fresh LB (with antibiotics) was added to 50 mL tubes, and the cultures were added to the fresh LB at 1 pm. Minipreps were made according to the [[Media:MODIFIED_Miniprep_Protocol.pdf|MODIFIED Miniprep]] at room temperature and stored in the 4ºC. |
- | + | ||
== 10/15/2010 == | == 10/15/2010 == | ||
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Incubation: | Incubation: | ||
- | * | + | *37ºC for 15 min |
- | * | + | *80ºC for 20 min |
Ligated according [http://www.roche-applied-science.com/pack-insert/4898117a.pdf Roche Rapid Ligation Protocol]. | Ligated according [http://www.roche-applied-science.com/pack-insert/4898117a.pdf Roche Rapid Ligation Protocol]. | ||
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Transformed according to [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]], but made 1:1 and 1:3 dilutions by adding 50 uL of cold, sterile DI water to the comp cells and mixing before each plating. | Transformed according to [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]], but made 1:1 and 1:3 dilutions by adding 50 uL of cold, sterile DI water to the comp cells and mixing before each plating. | ||
- | |||
== 10/16/2010 == | == 10/16/2010 == | ||
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[[Media:Quick Transformation of E. coli Competant Cell Preperation.pdf| Protocol - Competent Cell Preparation for Quick Transformation of E. coli]] | [[Media:Quick Transformation of E. coli Competant Cell Preperation.pdf| Protocol - Competent Cell Preparation for Quick Transformation of E. coli]] | ||
- | Ben | + | Ben started the overnight cultures and assisted in OD measurement and initial centrifugation steps. |
- | A colony of P. putida and P. fluorescence was picked from LB agar plates (10/6/10) and grown up in an overnight culture in 2ml Amp at | + | A colony of P. putida and P. fluorescence was picked from LB agar plates (10/6/10) and grown up in an overnight culture in 2ml Amp at 30ºC. |
- | The overnight culture was added to 100 ml of LB media and grown at | + | The overnight culture was added to 100 ml of LB media and grown at 30ºC for 4 hours. |
Protocol was carried out approximately 20 minutes after the following OD600 was recorded: | Protocol was carried out approximately 20 minutes after the following OD600 was recorded: | ||
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P. putida – 0.587 | P. putida – 0.587 | ||
- | |||
P. Fluorescence – 0.610 | P. Fluorescence – 0.610 | ||
- | One centrifuge tube containing p. putida culture failed during centrifugation at 5000 rpm. The protocol was altered by reducing the speed to 2500 rpm after this point to reduce the chance of failure. The cells are stored in the | + | One centrifuge tube containing p. putida culture failed during centrifugation at 5000 rpm. The protocol was altered by reducing the speed to 2500 rpm after this point to reduce the chance of failure. The cells are stored in the 4ºC fridge in room 1239. |
== 10/17/2010 == | == 10/17/2010 == | ||
''Marcus and Ben'' | ''Marcus and Ben'' | ||
- | Nanodropped Nick's miniprep of pSB1AT3-Flu (#8) from yesterday, but found low concentrations of about 13-18 ng/uL since each mL of overnight was miniprepped individually. The samples were pooled by using a repurification protocol in the miniprep kit, eluted in Ultrapure water, and | + | Nanodropped Nick's miniprep of pSB1AT3-Flu (#8) from yesterday, but found low concentrations of about 13-18 ng/uL since each mL of overnight was miniprepped individually. The samples were pooled by using a repurification protocol in the miniprep kit, eluted in Ultrapure water, and recived a final concentration of 40.2 ng/uL. The sequencing core requires 50 ng/uL, so a 5 mL overnight of pSB1AT3-Flu was made for Ben and Nick to miniprep tomorrow and a 2 mL overnight of DH5α for comp cells. |
== 10/18/2010 == | == 10/18/2010 == | ||
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''Marcus'' | ''Marcus'' | ||
- | Made 10 Tet and 10 Cm plates. Also, Nick and I transformed DH5alpha comp cells from - | + | Made 10 Tet and 10 Cm plates. Also, Nick and I transformed DH5alpha comp cells from -80ºC iGEM box with extra pSB1AT3-J23100-Flu ligation Ben and Kevin made yesterday. Transformed according to [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]]. We also transformed ''P. putida'' and ''P. fluorescens'' with pSB1AT3 as a test. No dilutions were made. It was noted while plating the cells that the ''Pseudomonas'' strains somehow wound up with less than 50 uL of cells. And due to the cells flocculating during storage, the ''Pseudomonas'' strains also had be vortexed to resuspend the cells. |
== 10/21/2010 == | == 10/21/2010 == | ||
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**VF2- 33.9 nmol/.05 nmol/uL= 678 uL | **VF2- 33.9 nmol/.05 nmol/uL= 678 uL | ||
**VR- 40 nmol/ .05 nmol/uL= 800 uL | **VR- 40 nmol/ .05 nmol/uL= 800 uL | ||
- | *Used a 2 min | + | *Used a 2 min extension |
- | Also, put the Cm and Tet plates from Tuesday in the | + | Also, put the Cm and Tet plates from Tuesday in the 4ºC. |
== 10/23/2010 == | == 10/23/2010 == | ||
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'''Expected Digest bands''' | '''Expected Digest bands''' | ||
- | *FimB- 638 bp | + | *FimB - 638 bp |
- | *VP130- 2376, 1925 bp | + | *VP130 - 2376, 1925 bp |
- | *pSB1AT3-Flu- 6770, 15 bp | + | *pSB1AT3-Flu - 6770, 15 bp |
The digests appear to have worked, although the ligations seem to have been incomplete (Roche Rapid Dephos & Ligation Kit). | The digests appear to have worked, although the ligations seem to have been incomplete (Roche Rapid Dephos & Ligation Kit). | ||
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- | 3:1 Insert:Vector VP130 | + | 3:1 Insert:Vector VP130: |
*.4 uL pSB1C3 | *.4 uL pSB1C3 | ||
*2.8 uL Vp130 | *2.8 uL Vp130 | ||
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*14.8 uL UP H2O | *14.8 uL UP H2O | ||
- | 3:1 Insert:Vector FimB | + | 3:1 Insert:Vector FimB: |
*.4 uL pSB1C3 | *.4 uL pSB1C3 | ||
*.9 uL FimB | *.9 uL FimB | ||
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*16.7 uL UP H2O | *16.7 uL UP H2O | ||
- | We then did the following transformations using the [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]] and the | + | We then did the following transformations using the [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]] and the DH5α comp cells Nick made using his [[Media:Quick Transformation of E. coli Competant Cell Preperation.pdf| Competent Cell Preparation for Quick Transformation of E. coli]] protocol: |
- | Transformation (Plate Resistance) | + | Transformation (Plate Resistance): |
*Cell only control (Cm) | *Cell only control (Cm) | ||
*9A pos control (Cm) | *9A pos control (Cm) | ||
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We suspect that the "linearized" backbone pSB1C3 provided in the 2010 distribution is not actually digested and the cause of our transformation failures. | We suspect that the "linearized" backbone pSB1C3 provided in the 2010 distribution is not actually digested and the cause of our transformation failures. | ||
- | Digest Mix | + | Digest Mix: |
*20 uL pSB1C3 (500 ng) | *20 uL pSB1C3 (500 ng) | ||
*5 uL NEB Buffer 2 | *5 uL NEB Buffer 2 | ||
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*23.5 uL Ultrapure water | *23.5 uL Ultrapure water | ||
- | Incubation | + | Incubation: |
*15 min @ 37C | *15 min @ 37C | ||
*20 min @ 80C | *20 min @ 80C | ||
Ligation: | Ligation: | ||
- | *pSB1C3- 4.572 fmol/uL | + | *pSB1C3 - 4.572 fmol/uL |
- | *VP130- 8.065 fmol/uL | + | *VP130 - 8.065 fmol/uL |
- | *FimB- 24.2 fmol/uL | + | *FimB - 24.2 fmol/uL |
- | VP130 Ligation Mix | + | VP130 Ligation Mix: |
*1 uL pSB1C3 | *1 uL pSB1C3 | ||
*2.8 uL VP130 | *2.8 uL VP130 | ||
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*13.2 uL Ultrapure water | *13.2 uL Ultrapure water | ||
- | FimB Ligation Mix | + | FimB Ligation Mix: |
*1 uL pSB1C3 | *1 uL pSB1C3 | ||
*0.9 uL FimB | *0.9 uL FimB | ||
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*15.1 uL Ultrapure water | *15.1 uL Ultrapure water | ||
- | Incubation | + | Incubation: |
- | *10 mins @ 22. | + | *10 mins @ 22.5ºC |
- | *10 mins @ | + | *10 mins @ 65ºC |
Ligations were transformed according to [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]]. | Ligations were transformed according to [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]]. |
Latest revision as of 01:08, 27 October 2010