Team:Michigan/Oil Sands August September

From 2010.igem.org

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[[Team:Michigan/Oil_Sands | Back to Oil Sands Main Notebook]]
==8/1/2010==
==8/1/2010==
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'''Nanodrop of pBAD and GFP verification'''
'''Nanodrop of pBAD and GFP verification'''
-
The miniprep was performed for the two cultures started last night according the the MODIFIED miniprep protocol.  A frozen stock was made of the pBAD biobrick and placed in the -80C freezer
+
This miniprep was performed for the two cultures started last night according the the MODIFIED miniprep protocol.  A frozen stock was made of the pBAD biobrick and placed in the -80°C freezer.
-
The DNA concentrations reported by the nanodrop are as follows
+
The DNA concentrations reported by the nanodrop are as follows:
*pBAD: 386 ng/uL (260/280=2.02 and 260/230=2.32)
*pBAD: 386 ng/uL (260/280=2.02 and 260/230=2.32)
*GFP: 25.8 ng/uL (260/280=2.22 and 260/230=2.72)
*GFP: 25.8 ng/uL (260/280=2.22 and 260/230=2.72)
-
The pBAD grew out very dense and when IPTG is added the copy number is over 100 so the DNA concentration was very high
+
The pBAD grew out very densely; when IPTG is added, the copy number is over 100. The DNA concentration was very high.
'''Digest of pBAD and GFP verification'''
'''Digest of pBAD and GFP verification'''
-
The parts were digested according to the protocol in the protocol section of the wiki for a 50 uL reaction volume
+
The parts were digested according to the protocol in the protocol section of the wiki for a 50 uL reaction volume:
*GFP #3 was digested with XbaI and PstI
*GFP #3 was digested with XbaI and PstI
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[[Image:8-2-2010_gel_of_pbad_and_gfp_annotated.jpg|400px]]
[[Image:8-2-2010_gel_of_pbad_and_gfp_annotated.jpg|400px]]
-
*Lane 1: invitrogen 1 kb plus ladder
+
*Lane 1: Invitrogen 1 kb Plus ladder
-
*Lane 2: GFP #3 cut with XbaI and PstI (GFP: 720 bp Backbone: 2079 bp)
+
*Lane 2: GFP #3 cut with XbaI and PstI (GFP: 720 bp; Backbone: 2079 bp)
*Lane 3: GFP #4 cut with EcoRI and XbaI
*Lane 3: GFP #4 cut with EcoRI and XbaI
*Lane 4: uncut GFP plamid (only 1 uL left so did not show up on gel)
*Lane 4: uncut GFP plamid (only 1 uL left so did not show up on gel)
-
*Lane 5: pBAD cut with EcoRI and SpeI(pBAD: 1210 bp backbone: 4425 bp)
+
*Lane 5: pBAD cut with EcoRI and SpeI (pBAD: 1210 bp; Backbone: 4425 bp)
*Lane 6: pBAD cut with SpeI and PstI
*Lane 6: pBAD cut with SpeI and PstI
*Lane 7: uncut pBAD (loaded very small amount into gel by accident)
*Lane 7: uncut pBAD (loaded very small amount into gel by accident)
*Lane 8: uncut pBAD (loaded 1 uL of miniprep into gel)
*Lane 8: uncut pBAD (loaded 1 uL of miniprep into gel)
-
We did not get the pBAD part potentially because the miniprep culture overgrew and the cells started to lyse their chromosomal DNA (resulting in extremely long bands in the gel).  We will try miniprepping the DNA from this culture again letting the culture grow out for a shorter amount of time before the miniprep
+
We did not get the pBAD part due to the miniprep culture overgrowing and the cells lysing their chromosomal DNA (resulting in extremely long bands in the gel).  We will try miniprepping the DNA from this culture again letting the culture grow out for a shorter amount of time before the miniprep.
'''flu primers'''
'''flu primers'''
-
Primers were ordered today to amplify the flu operon of E. coli K12 by colony PCR
+
Primers were ordered today to amplify the flu operon of E. coli K12 by colony PCR:
*[[Media:7-31-2010_Flu_operon_primers.pdf|General primer design]]
*[[Media:7-31-2010_Flu_operon_primers.pdf|General primer design]]
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==8/3/2010==
==8/3/2010==
-
'''Minimal Media Recipes'''
+
'''Minimal Media'''
-
Minimal media was mixed today according to the following [[Media:8-3-2010_media_recipies_updated.pdf|recipes]] (also found in the media recipe section of the notebooks)
+
Minimal media was mixed today according to the following [[Media:8-3-2010_media_recipies_updated.pdf|recipes]] (also found in the media recipe section of the notebooks):
-
'''Culturing Bacteria in Naphthenic Acids (NA's)'''
+
'''Culturing Bacteria in Naphthenic Acids (NAs)'''
-
The following 9 cultures were started to test bacteria growth in NA media
+
The following 9 cultures were started to test bacteria growth in NA media:
-
*Negitive Control (to make sure all strains grow in this minimal media)
+
*Negative Control (to make sure all strains grow in this minimal media)
**E. coli K12 in Bushnell-Haas minimal media supplemented with glucose
**E. coli K12 in Bushnell-Haas minimal media supplemented with glucose
-
**P. putida oil sands in Bushnell-Haas minimal media supplemented with glucose
+
**P. putida oilsands in Bushnell-Haas minimal media supplemented with glucose
-
**P. flourescens oil sands in Bushnell-Haas minimal media supplemented with glucose
+
**P. flourescens oilsands in Bushnell-Haas minimal media supplemented with glucose
*NA media
*NA media
**E. coli K12 in Bushnell-Haas minimal media supplemented with cyclohexane carboxylic acid (120 mg/L concentration)
**E. coli K12 in Bushnell-Haas minimal media supplemented with cyclohexane carboxylic acid (120 mg/L concentration)
-
**P. putida oil sands in Bushnell-Haas minimal media supplemented with cyclohexane carboxylic acid (120 mg/L concentration)
+
**P. putida oilsands in Bushnell-Haas minimal media supplemented with cyclohexane carboxylic acid (120 mg/L concentration)
-
**P. flourescens oil sands in Bushnell-Haas minimal media supplemented with cyclohexane carboxylic acid (120 mg/L concentration)
+
**P. flourescens oilsands in Bushnell-Haas minimal media supplemented with cyclohexane carboxylic acid (120 mg/L concentration)
*pH adjusted NA media
*pH adjusted NA media
-
**E. coli K12 in Bushnell-Haas minimal media supplemented with cyclohexane carboxylic acid (120 mg/L concentration) pH adjusted between 8 and 9
+
**E. coli K12 in Bushnell-Haas minimal media supplemented with cyclohexane carboxylic acid (120 mg/L concentration), pH adjusted between 8 and 9
-
**P. putida oil sands in Bushnell-Haas minimal media supplemented with cyclohexane carboxylic acid (120 mg/L concentration)pH adjusted between 8 and 9
+
**P. putida oilsands in Bushnell-Haas minimal media supplemented with cyclohexane carboxylic acid (120 mg/L concentration), pH adjusted between 8 and 9
-
**P. flourescens oil sands in Bushnell-Haas minimal media supplemented with cyclohexane carboxylic acid (120 mg/L concentration)pH adjusted between 8 and 9
+
**P. flourescens oilsands in Bushnell-Haas media supplemented with cyclohexane carboxylic acid (120 mg/L concentration), pH adjusted between 8 and 9
-
1 mL of the above media was added to a 15 mL falcon tube and innoculated with -80C freezer stock of each culture and grown in the 30C shaker (psuedomonas strains are temperature sensitive).  The cultures were started at 7:30 pm
+
1 mL of the above media was added to a 15 mL falcon tube and inoculated with -80°C freezer stock of each culture and grown in the 30C shaker (Psuedomonas strains are temperature sensitive).  The cultures were started at 7:30 pm.
==8/4/2010==
==8/4/2010==
-
'''Culturing Bacteria in Naphthenic Acids (NA's)'''
+
'''Culturing Bacteria in Naphthenic Acids (NAs)'''
-
At 12:30 pm the OD600 of the cultures started yesterday were measured and are listed below.  These results must be taken with a grain of salt because the cultures were started from the -80C freezer stock with different amounts of innoculum
+
At 12:30 pm the OD600 of the cultures started yesterday were measured and are listed below.  These results must be taken with a grain of salt, because the cultures were started from the -80°C freezer stock with different amounts of inoculum:
*P. putida BH-glu: 0.244
*P. putida BH-glu: 0.244
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*E. coli K12 BH-CHCA pH9: 0.0325
*E. coli K12 BH-CHCA pH9: 0.0325
-
P. flourescens oilsand seems to love this medium and grew out very dense! P. putida oilsand showed signs of starting to grow. E. coil K12 did not grow at all and dead cells pellets appeared on the bottom of the culture flask
+
P. flourescens oilsands seems to love this medium and grew out very densely! P. putida (oilsands) showed signs of growing. E. coli K12 did not grow at all and pellets of dead cells appeared on the bottom of the culture flask.
-
From these results we can go ahead and start cultures for the biofilm assay experiment, and time the cultures so they all become dense on around the same day.  If all cultures are started 24 hours in advanced they all should saturate by the next day.
+
From these results we can start cultures for the biofilm assay experiment and time the cultures so they all become dense around the same day.  If all cultures are started 24 hours in advance they all should saturate by the next day.
-
The E. coli K12 and P. putida oilsands cultures were placed back in the 30C shaker to keep growing.  By 9:30pm the P. putida oilsands cultures had saturated and the E. coli K12 cultures had still not grown
+
The E. coli K12 and P. putida oilsands cultures were placed back in the 30°C shaker to keep growing.  By 9:30pm, the P. putida oilsands cultures had saturated and the E. coli K12 cultures had still not grown.
As a control for the biofilm assay E. coli K12 will be grown in M9 minimal media with glucose as a carbon source with and without casamino acids.  We will not attempt to grow E. coli K12 in Bushnell-Haas media.   
As a control for the biofilm assay E. coli K12 will be grown in M9 minimal media with glucose as a carbon source with and without casamino acids.  We will not attempt to grow E. coli K12 in Bushnell-Haas media.   
-
'''Measuring pH of Bushnell-Haas minial media with cyclohexane carboxylic acid adjusted to pH 9'''
 
-
Today Marc showed us how to use the pH meter in the Lin Lab.  Yesterday the pH of the media was estimated by pH paper to be between 8 and 9.  The actual pH of the media is 7.35.  The pH of the already adjusted media from yesterday was increased by added more 250 uL of 0.1 M NaOH to 5mL of BH CHCA media and the pH ready 7.6 using the pH probe.  Another 250 uL of NaOH was added to this mixutre and the pH read 8.8 using the pH probe.  pH paper was dotted using this mixture to see the exact color it should turn when the pH is close to 9 (the paper looks more blue than green when the dot first appears). 
+
'''Measuring pH of Bushnell-Haas minimal media with cyclohexane carboxylic acid adjusted to pH 9'''
 +
Today Marc showed us how to use the pH meter in the Lin Lab.  Yesterday the pH of the media was estimated by pH paper to be between 8 and 9.  The actual pH of the media is 7.35.  The pH of the already adjusted media from yesterday was increased by added more 250 uL of 0.1 M NaOH to 5mL of BH-CHCA media and the pH read 7.6 using the pH probe.  Another 250 uL of NaOH was added to this mixture and the pH read 8.8 using the pH probe.  pH paper was dotted using this mixture to see the exact color it should turn when the pH is close to 9 (the paper looks more blue than green when the dot first appears). 
A more concentrated NaOH sterilized stock solution needs to be made so when NaOH is added to the media the salts are not diluted
A more concentrated NaOH sterilized stock solution needs to be made so when NaOH is added to the media the salts are not diluted
-
'''Bacterial Growth in pH of 9'''
 
-
After determining that 1mL of the supposedly adready adjusted BH-CHCA media needed 100 uL of 0.1 M NaOH soultion per mL to really be close to a pH of 9, new overnight cultures were started of the two pseudomonas oilsand strains in the 1 mL of the BH-CHCA pH adjusted media from yesterday adding an additional 100 uL of 0.1 M NaOH.  These two cultures were placed in the 30C shaker to grow out overnight at 8:30 pm. 
+
'''Bacterial Growth in pH 9'''
 +
After determining that 1 mL of the supposedly already adjusted BH-CHCA media needed 100 uL of 0.1 M NaOH solution per mL to really be close to a pH of 9, new overnight cultures were started of the two Pseudomonas oilsands strains in 1 mL of the BH-CHCA pH-adjusted media from yesterday with an additional 100 uL of 0.1 M NaOH added.  These two cultures were placed in the 30°C shaker to grow out overnight at 8:30 pm. 
-
'''Miniprep of pBAD take 2'''
 
-
At 10:45 pm a 5 mL culture of the pBAD biobrick was started from the -80C freezer stock in LB + 25 mg/mL KAN + 1 mM IPTG for a miniprep tomorrow morning
+
'''Miniprep of pBAD, take 2'''
 +
 
 +
At 10:45 pm a 5 mL culture of the pBAD biobrick was started from the -80°C freezer stock in LB + 25 mg/mL KAN + 1 mM IPTG for a miniprep tomorrow morning.
'''PCR of flu operon of E. coli K12'''
'''PCR of flu operon of E. coli K12'''
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The primers ordered on Monday came in today!
The primers ordered on Monday came in today!
-
PCR reagents were picked up from the life science store
+
PCR reagents were picked up from the life science store.
-
*dNTP's
+
*dNTPs
-
**These dNTP's came in four seperate containers individually in a 100 mM solution.  The following recipe was used to make the 10 mM dNTP mixture
+
**These dNTPs came in four separate containers individually in a 100 mM solution.  The following recipe was used to make the 10 mM dNTP mixture:
***100 uL of 100 mM dATP
***100 uL of 100 mM dATP
***100 uL of 100 mM dTTP
***100 uL of 100 mM dTTP
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***100 uL of 100 mM dCTP
***100 uL of 100 mM dCTP
***600 uL of ultra pure water
***600 uL of ultra pure water
-
*Phusion High Fidelity Polymerase
+
*Phusion High Fidelity Polymerase:
**comes with buffer
**comes with buffer
**used to amplify long pieces of DNA
**used to amplify long pieces of DNA
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The PCR was run according to the following [[Media:8-4-2010_Colony_PCR_flu.pdf|colony PCR protocol]]
The PCR was run according to the following [[Media:8-4-2010_Colony_PCR_flu.pdf|colony PCR protocol]]
-
The final annealing cycle for 10 minutes at 72 C was accidentally added in the last 30 cycle loop.  This mistake was caught after 3 cycles and the PCR was stopped, the program fixed and the reaction restarted.   
+
The final annealing cycle for 10 minutes at 72°C was accidentally added in the last 30 cycle loop.  This mistake was caught after 3 cycles and the PCR was stopped, the program fixed and the reaction restarted.   
'''Gel of flu colony PCR'''
'''Gel of flu colony PCR'''
-
The following gel was run in a 0.7% agarose gel at 85V according to the protocol on the wiki
+
The following gel was run in a 0.7% agarose gel at 85 V according to the protocol on the wiki:
[[Image:8-4-2010_PCR_of_flu_operon.jpg|500px]]
[[Image:8-4-2010_PCR_of_flu_operon.jpg|500px]]
-
For the first 5 cycles the annealing temperature was lowered to the Tm of the 16 or 18 bp that was complementary to the E. coli K12 template.  A gradient of temperature was run for the annealing temperatures of the first five cylces as labeled in the gel
+
For the first 5 cycles the annealing temperature was lowered to the Tm of the 16 or 18 bp that was complementary to the E. coli K12 template.  A gradient of temperature was run for the annealing temperatures of the first five cycles as labeled in the gel.
==8/5/2010==
==8/5/2010==
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'''Bacterial Growth at pH 9'''
'''Bacterial Growth at pH 9'''
-
At 9:00am the two cultures started in the BH-CHCA with the pH actually adjusted to around 9 was checked.  Both P. putida oilsands and P. fluorescens oilsands showed slight signs of growth.  Later in the day at3:30 pm the cultures are denser were denser and the OD600 of the culture is:
+
At 9:00am the two cultures started in the BH-CHCA were tested to ensure the pH was around 9.  Both P. putida oilsands and P. fluorescens oilsands showed slight signs of growth.  Later, in the day at 3:30pm, the cultures were denser; the OD600 of the culture was:
*P. putida oilsands: 0.065
*P. putida oilsands: 0.065
*P. fluorescens oilsands: 0.433
*P. fluorescens oilsands: 0.433
-
It should be noted some of the salts precipitated out of solution in the 1 mL cultures
+
It should be noted some of the salts precipitated out of solution in the 1 mL cultures.
-
'''Miniprep of pBAD take 2'''
+
'''Miniprep of pBAD, take 2'''
-
The miniprep was started at 8:45am.  After 10 hours of growth the culture was saturated and the miniprep started according to the modified miniprep protocol.
+
The miniprep was started at 8:45am.  After 10 hours of growth the culture was saturated and the miniprep initialized according to the modified miniprep protocol.
-
'''Nanodrop of pBAD take 2'''
+
'''Nanodrop of pBAD, take 2'''
-
The concentration of the pBAD promoter miniprepped earlier today is 418 ng/uL.  It is most likely still contaminated with this high concentration but we will digest it and run a gel to check anyways
+
The concentration of the pBAD promoter miniprepped earlier today is 418 ng/uL.  It is most likely still contaminated with this high concentration but we will still digest it and run a gel to check.
-
'''Digest of pBAD take 2'''
+
'''Digest of pBAD, take 2'''
-
the digest was performed according to the digest protocol in the protocol section of the wiki
+
This digest was performed according to the digest protocol in the protocol section of the wiki:
pBAD #3 was digested with EcoRI and SpeI
pBAD #3 was digested with EcoRI and SpeI
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'''PCR of flu operon'''
'''PCR of flu operon'''
-
The PCR of the flu operon was repeated 3 more times at higher annealing temperatures because this showed to have less unspecific product (shown by the gel from yesterday)according to this [[Media:8-5-2010_Colony_PCR_flu.pdf|modified colony PCR protocol]].  The volume of the reactions were increased to 50 uL to do a PCR purification step afterwards.
+
The PCR of the flu operon was repeated 3 more times at higher annealing temperatures as this showed to have less unspecific product (shown by the gel from yesterday) according to this: [[Media:8-5-2010_Colony_PCR_flu.pdf|modified colony PCR protocol]].  The volumes of the reactions were increased to 50 uL for a PCR purification step afterwards.
'''Gel of flu operon PCR and pBAD promoter digest'''
'''Gel of flu operon PCR and pBAD promoter digest'''
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The gel ran exactly the same for the pBAD promoter as on 8/2/2010.  The sample is probably contaminated and a new colony should be picked from the transformation plate and screened.   
The gel ran exactly the same for the pBAD promoter as on 8/2/2010.  The sample is probably contaminated and a new colony should be picked from the transformation plate and screened.   
-
Also, no bands appeared for the flu PCR.  One error might be because we did not include an initial cell lysis step.  The PCR will be tried again tomorrow
+
Also, no bands appeared for the flu PCR.  One error might be because we did not include an initial cell lysis step.  The PCR will be attempted again tomorrow.
'''Biofilm assay in minimal media'''
'''Biofilm assay in minimal media'''
-
Cultures were started according to the [[Media:8-5-2010_Biofilm_Formation_Experiment.pdf|following biofilm assay protocol]] to inoculate the microplate for the biofilm assay tomorrow.
+
Cultures were started according to the [[Media:8-5-2010_Biofilm_Formation_Experiment.pdf|biofilm assay protocol]] for the inoculation of the microplate; these will be used in the biofilm assay tomorrow.
==8/6/2010==
==8/6/2010==
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'''PCR of flu take 3'''
'''PCR of flu take 3'''
-
The first PCR of the flu operon was repeated with an added cell lysis step according to the [[Media:8-6-2010_Colony_PCR_flu.pdf|following modified protocol]]
+
The first PCR of the flu operon was repeated with an added cell lysis step according to the [[Media:8-6-2010_Colony_PCR_flu.pdf|following modified protocol]]:
'''Gel of PCR of flu take 3'''
'''Gel of PCR of flu take 3'''
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Invitrogen 1 kb plus ladder
Invitrogen 1 kb plus ladder
-
Due to the unspecific product the flu operon will be purified with gel purification
+
Due to the unspecific product the flu operon will be purified with gel purification.
'''Biofilm Assay'''
'''Biofilm Assay'''
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|}
-
Not all of the cultures grew out (P. putida KT2440 Bushnell-Haas media plus cyclohexane carboylic acid, P. putida KT2440 Bushnell-Haas media plus cyclohexane carboylic acid pH 9, P. putida oilsands Bushnell-Haas media plus cyclohexane carboxylic acid pH 9) The [[Media:8-6-2010_Biofilm_Formation_Experiment.pdf|modified bio assay protocol]]
+
Not all of the cultures grew out (P. putida KT2440 Bushnell-Haas media plus cyclohexane carboylic acid, P. putida KT2440 Bushnell-Haas media plus cyclohexane carboylic acid pH 9, P. putida oilsands Bushnell-Haas media plus cyclohexane carboxylic acid pH 9). The [[Media:8-6-2010_Biofilm_Formation_Experiment.pdf|modified bio assay protocol]]
-
was used to inoculate the microplate
+
was used to inoculate the microplate.
-
The microplate was inoculated at 4:30pm
+
The microplate was inoculated at 4:30pm.
==8/8/2010==
==8/8/2010==
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'''Biofilm Assay'''
'''Biofilm Assay'''
-
The washing and CV staining of the bioassay was performed according to the modified protocol on 8/6/2010
+
The washing and CV staining of the bioassay was performed according to the modified protocol on 8/6/2010.
-
The OD 600 of the plate after the bioassay growth is shown in the following graph
+
The OD 600 of the plate after the bioassay growth is shown in the following graph:
[[Image:8-8-2010_OD_600_after_biofilm_growth_graph.jpg|500px]]
[[Image:8-8-2010_OD_600_after_biofilm_growth_graph.jpg|500px]]
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-
The Crystal Violet OD 600 after staining is show in the following graph
+
The Crystal Violet OD 600 after staining is show in the following graph:
[[Image:8-8-2010_CV_OD_600_biofilm_after_staining.jpg|500px]]
[[Image:8-8-2010_CV_OD_600_biofilm_after_staining.jpg|500px]]
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-
The Crystal Violet OD 600 to OD 600 after growth ratio is shown in the following graph
+
The Crystal Violet OD 600 to OD 600 after growth ratio is shown in the following graph:
[[Image:8-8-2010_CV_OD_600_to_OD_600_ratio_graph.jpg|500px]]
[[Image:8-8-2010_CV_OD_600_to_OD_600_ratio_graph.jpg|500px]]
-
The negitive control (E. coli K12 in M9 minimal media with glucose) grew one of the best biofilms contradicting what was expected.  From here we need to find a knock out strain that is deficent in forming biofilms.  
+
The negative control (E. coli K12 in M9 minimal media with glucose) grew one of the best biofilms; this contradicted what was expected.  From here we need to find a knock out strain that is deficient in forming biofilms.  
-
A strong conclusion cannot be drawn about the biofilm forming abilities in minimal media with cyclohexane carboxylic acids (CHCA) because the cells did not grow out very dense.  This is why the error bars are large.
+
A strong conclusion cannot be drawn about the biofilm forming abilities in minimal media with cyclohexane carboxylic acids (CHCA) as the cells did not grow very densely.  This is the reason for the large error bars.
-
It is also interesting that P. fluorescens in pH 9 did grow out but did not form biofilms even though the cell culture grew relatively well.  We need a negitive control though to confirm this result
+
It is interesting that P. fluorescens in pH 9 did not form biofilms, even though the cell culture grew relatively well.  We need a negative control to confirm this result.
==8/9/2010==
==8/9/2010==
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'''PCR of flu for gel extraction'''
'''PCR of flu for gel extraction'''
-
Because there are unspecific products from the PCR of flu, a gel extraction will have to be used to isolate the desired DNA
+
Because there are unspecific products from the PCR of flu, a gel extraction will have to be used to isolate the desired DNA.
-
50 uL reaction volume for PCR was performed using 65.5C for the initial annealing temperature as shown in the following [[Media:8-9-2010_Colony_PCR_flu.pdf|colony PCR protocol of flu]].  The higher annealing temperature reduces the unspecific products
+
50 uL reaction volume for PCR was performed using 65.5C for the initial annealing temperature as shown in the following [[Media:8-9-2010_Colony_PCR_flu.pdf|colony PCR protocol for flu]].  The higher annealing temperature reduces unspecific products.
'''Gel Extraction of flu operon'''
'''Gel Extraction of flu operon'''
-
A gel extraction was performed according to the following [[Media:8-8-2010_Protocol_for_gel_extraction.pdf|gel extraction protocol]]
+
A gel extraction was performed according to the following [[Media:8-8-2010_Protocol_for_gel_extraction.pdf|gel extraction protocol]].
-
20 uL of the flu #14 PCR was mixed with 5 uL of blue juice to run the gel
+
20 uL of the flu #14 PCR was mixed with 5 uL of blue juice to run the gel.
-
The weight of the eppendorf tube was 1.0284 g
+
The weight of the eppendorf tube was 1.0284 g.
-
The weight of the eppendorf tube with the sample was 1.2030 g
+
The weight of the eppendorf tube with the sample was 1.2030 g.
-
The weight of the gel sample was 0.175 g
+
The weight of the gel sample was 0.175 g.
-
524 uL of QG buffer was added to disolve the gel sample
+
524 uL of QG buffer was added to dissolve the gel sample.
-
No DNA appeared on the nanodrop, Marc ran the flu gel purified sample on his gel to confirm no DNA eluted
+
No DNA appeared on the nanodrop. Marc ran the flu gel purified sample on his gel to confirm no DNA eluted.
-
DNA did appear on the gel that Marc ran so we do have DNA from the gel extraction.  The nanodrop may not have been able to pick it up because it was too low of a concentration or it was contaminated (very high 260/230 reading)
+
DNA did appear on the gel that Marc ran so we do have DNA from the gel extraction.  The nanodrop may not have been able to pick it up because it was too low of a concentration or it was contaminated (very high 260/230 reading).
[[Image:8-9-2010_Gel_extraction_of_flu.jpg|500px]]
[[Image:8-9-2010_Gel_extraction_of_flu.jpg|500px]]
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''Ann and Bryce''
''Ann and Bryce''
-
'''Transformation of Constituative Promoter and KAN backbone'''
+
'''Transformation of Constitutive Promoter and KAN backbone'''
-
The transformation was performed according to the electroporation protocol in the protocol section of the wiki
+
The transformation was performed according to the electroporation protocol in the protocol section of the wiki.
-
The comp cell culture was started at 9:10am by adding the 8 mL overnight culture into 40mL of LB media in a 500 mL sterile flask
+
The comp cell culture was started at 9:10am by adding the 8 mL overnight culture into 40mL of LB media in a 500 mL sterile flask.
-
The OD600 of the culture was checked at 11:20am and was 1.0.  This culture was slightly overgrown but was immediately placed on ice and used anyways (this might lower the efficency of the procedure).   
+
The OD600 of the culture was checked at 11:20am and was 1.0.  This culture was slightly overgrown but was immediately placed on ice and used anyways (this might lower the efficiency of the procedure).   
The biobricks we are transforming today are:
The biobricks we are transforming today are:
-
*J23100 (constituative promoter)
+
*J23100 (constitutive promoter)
-
*I14018 (PCR purify to remove protomoter and be left with backbone that has KAN resistance)
+
*I14018 (PCR purify to remove promoter and be left with backbone that has KAN resistance)
*(pBAD biobrick taken from the registry as a positive control)
*(pBAD biobrick taken from the registry as a positive control)
-
The transformation was done at 2:15pm and the cells were plated on the following plates
+
The transformation was done at 2:15pm and the cells were plated on the following plates:
-
*negitive control (comp cells only) on LB+100 mg/mL AMP
+
*negative control (comp cells only) on LB+100 mg/mL AMP
-
*constituative promoter on LB+100 mg/mL AMP
+
*constitutive promoter on LB+100 mg/mL AMP
-
*negitive control (comp cells only) on LB+25 mg/mL KAN + 1mM IPTG
+
*negative control (comp cells only) on LB+25 mg/mL KAN + 1mM IPTG
*positive control (pBAD promoter taken from the registry) on LB+25 mg/mL KAN + 1mM IPTG
*positive control (pBAD promoter taken from the registry) on LB+25 mg/mL KAN + 1mM IPTG
*backbone on LB+25 mg/mL KAN + 1mM IPTG
*backbone on LB+25 mg/mL KAN + 1mM IPTG
-
and placed in the 37C incubator to grow out overnight
+
The plates were placed in the 37°C incubator to grow out overnight.
-
We plan on ligating the flu operon into the KAN backbone than cutting than inserting the promoter in front of this part.  We have to take this approach because the flu operon cannot be digested with PstI.
+
We plan on ligating the flu operon into the KAN backbone than cutting than inserting the promoter in front of this part.  We must take this approach because the flu operon cannot be digested with PstI.
----
----
Line 476: Line 479:
**51.3 g 1M NaOH
**51.3 g 1M NaOH
**38.6 g ore
**38.6 g ore
-
*Put beaker in shaker for 48 hours
+
*Put beaker in shaker for 48 hours
See the [[Media:NA Extraction.pdf|NA Extraction Protocol]].
See the [[Media:NA Extraction.pdf|NA Extraction Protocol]].
Line 484: Line 487:
'''Miniprep of constitutive promoter and backbone with KAN resistance'''
'''Miniprep of constitutive promoter and backbone with KAN resistance'''
-
The transformation was successful and both plates with the constitutive promoter and the backbone with KAN resistance grew up (even though the positive control did not grow...)
+
The transformation was successful; both plates with the constitutive promoter and the backbone with KAN resistance grew up (even though the positive control did not grow...).
The constitutive promoter part is followed by RFP.  We saw this on the transformation plates because the colonies were red.   
The constitutive promoter part is followed by RFP.  We saw this on the transformation plates because the colonies were red.   
Line 492: Line 495:
*constitutive promoter: LB + 100 mg/mL AMP
*constitutive promoter: LB + 100 mg/mL AMP
-
These cultures were placed in the 37C incubator to grow up
+
These cultures were placed in the 37°C incubator to grow up.
-
at 9:00pm the cultures were minipreped according to the modified miniprep protocol
+
At 9:00pm, the cultures were miniprepped according to the modified miniprep protocol.
'''Nanodrop of constitutive promoter and KAN backbone'''
'''Nanodrop of constitutive promoter and KAN backbone'''
Line 504: Line 507:
'''PCR of flu operon from the gel extraction product'''
'''PCR of flu operon from the gel extraction product'''
-
Using gel extraction product for ligations lowers the efficency of the ligation.  To get around this problem we performed another PCR reaction using the gel extraction product as the template according to the following [[Media:8-11-2010_Colony_PCR_flu.pdf|PCR protocol]]
+
Using gel extraction product for ligations lowers the efficiency of the ligation.  To get around this problem we performed another PCR reaction using the gel extraction product as the template according to the following [[Media:8-11-2010_Colony_PCR_flu.pdf|PCR protocol]].
'''Gel of PCR of flu operon from gel extraction product'''
'''Gel of PCR of flu operon from gel extraction product'''
-
There was more unspecific product from trying the PCR this reaction from the gel extracted part, so the gel extracted part will be used for further genetic manipulations
+
There was more unspecific product from trying the PCR this reaction from the gel extracted part, so the gel extracted part will be used for further genetic manipulations.
==8/12/2010==
==8/12/2010==
Line 514: Line 517:
'''Digest of constitutive promoter, KAN backbone and flu operon'''
'''Digest of constitutive promoter, KAN backbone and flu operon'''
-
The following digests were performed
+
The following digests were performed:
*constitutive promoter: EcoRI and SpeI (labeled P ES)
*constitutive promoter: EcoRI and SpeI (labeled P ES)
*constitutive promoter: EcoRI and XbaI (labeled P EX)
*constitutive promoter: EcoRI and XbaI (labeled P EX)
Line 523: Line 526:
All digests except for the KAN backbone digested with EcoRI and SpeI were 20 uL volumes.  1 uL of each enzyme was added along with 0.5 uL of BSA to the 20 uL reactions and the rest of the reagents were ajusted by multiplying the 50 uL volume by 0.4.  To make up for the extra volume of BSA and enzymes, the extra amount was subtracted from the amount of water added.   
All digests except for the KAN backbone digested with EcoRI and SpeI were 20 uL volumes.  1 uL of each enzyme was added along with 0.5 uL of BSA to the 20 uL reactions and the rest of the reagents were ajusted by multiplying the 50 uL volume by 0.4.  To make up for the extra volume of BSA and enzymes, the extra amount was subtracted from the amount of water added.   
-
Because the KAN backbone digested with EcoRI and SpeI is going to be PCR purified, double the amount of DNA was added and a 50 uL volume reaction was performed
+
Because the KAN backbone digested with EcoRI and SpeI is going to be PCR purified, double the amount of DNA was added and a 50 uL volume reaction was performed.
-
Since no nanodrop reading was made for the flu operon, no water was added and only the gel extraction was used
+
Since no nanodrop reading was made for the flu operon, no water was added and only the gel extraction was used.
'''Gel of constitutive promoter, KAN backbone and flu operon'''
'''Gel of constitutive promoter, KAN backbone and flu operon'''
Line 531: Line 534:
[[Image:8-13-2010_get_of_flu_KAN_ligation.JPG|500px]]
[[Image:8-13-2010_get_of_flu_KAN_ligation.JPG|500px]]
-
All parts digested accordingly and are ready for the ligation
+
All parts digested accordingly and are ready for the ligation.
'''PCR purification of KAN backbone'''
'''PCR purification of KAN backbone'''
Line 537: Line 540:
The PCR purification was performed with the 50 uL digest of the KAN backbone with EcoRI and SpeI according to the protocol in the protocol section of the wiki.   
The PCR purification was performed with the 50 uL digest of the KAN backbone with EcoRI and SpeI according to the protocol in the protocol section of the wiki.   
-
250 uL of buffer PB was used to mix with the digest before adding the mixture to the column
+
250 uL of buffer PB was used to mix with the digest before adding the mixture to the column.
'''Nanodrop of PCR purified KAN backbone'''
'''Nanodrop of PCR purified KAN backbone'''
-
The PCR purified KAN backbone had a concentration of 14.8 ng/uL with a 260/280 ratio of 1.86 and a 260/230 ratio of 0.91.  These ratios indicate that the sample is contaminated but there is still a high enough peak at 260 to show DNA is there.  Another gel could be run to verify that DNA is there, but this step will be skipped and the DNA will be used for the ligation
+
The PCR purified KAN backbone had a concentration of 14.8 ng/uL with a 260/280 ratio of 1.86 and a 260/230 ratio of 0.91.  These ratios indicate that the sample is contaminated but there is still a high enough peak at 260 to show DNA is present.  Another gel could be run to verify this, but this step will be skipped and the DNA will be used for the ligation.
'''Ligation of flu operon with the KAN backbone'''
'''Ligation of flu operon with the KAN backbone'''
-
The ligation was performed according to the following [[Media:8-11-2010_Ligation_Calculations_for_flu_in_plasmid_pSB2K3.pdf|T4 ligase protocol]]
+
The ligation was performed according to the following [[Media:8-11-2010_Ligation_Calculations_for_flu_in_plasmid_pSB2K3.pdf|T4 ligase protocol]].
----
----
Line 551: Line 554:
''Marcus''
''Marcus''
-
Removed ore/NaOH extraction mixture from shaker. Poured liquid into 50 mL tube, and strained the solids through a coffee filter and funnel to collect as much liquid as possible. Recovered about 20 mL. This was filtered through a 50 mL Steriflip vacuum tube, however, the filter became clogged and a second one was used, which required an extended vacuum time (in the future the mixture should be centrifuged first for several minutes). Recovered a total of about 40 mL (???).
+
Removed ore/NaOH extraction mixture from shaker. Poured liquid into 50 mL tube, and strained the solids through a coffee filter and funnel to collect as much liquid as possible; 20mL was recovered. This was filtered through a 50 mL Steriflip vacuum tube; however, the filter became clogged and a second one was used. This required an extended vacuum time (in the future the mixture should be centrifuged first for several minutes). Recovered a total of about 40 mL (???).
== 8/13/2010 ==
== 8/13/2010 ==
''Marcus''
''Marcus''
-
*Autoclaved 1.5 mL tubes
+
*Autoclaved 1.5 mL tubes.
-
*Took two bags of autoclave waste to DOW building to be autoclaved
+
*Took two bags of autoclave waste to DOW building to be autoclaved.
-
 
+
==8/14/2010==
==8/14/2010==
Line 564: Line 566:
'''Transformation of the Flu Operon + Kan backbone ligation'''
'''Transformation of the Flu Operon + Kan backbone ligation'''
-
The [[Media:Protocol_Heat_Shock_Transformation_2.pdf|Heat shock transformation protocol]] was used
+
The [[Media:Protocol_Heat_Shock_Transformation_2.pdf|Heat shock transformation protocol]] was used.
-
''E.Coli DH5alpha'' was used as competent cells and had a prewashing OD600 of .845 and a post washing OD600 of .787.
+
''E.Coli DH5α'' was used as competent cells and had a prewashing OD600 of .845 and a post washing OD600 of .787.
-
Two seperate transformations were perfromed both with 100 uL Comp. Cells + 10 uL Flu + KAN BB Ligation
+
Two separate transformations were performed both with 100 uL Comp. Cells + 10 uL Flu + KAN BB Ligation.
Both transformations were plated on LB+Kan+IPTG(30 uL) and are currently incubating at 37 C in the ERB.
Both transformations were plated on LB+Kan+IPTG(30 uL) and are currently incubating at 37 C in the ERB.
Line 580: Line 582:
*The second transformation plate showed minimal colonies.  
*The second transformation plate showed minimal colonies.  
-
Both plates were left to incubate longer because the slow growth may be due to the high concentration of Kanamycin (50mg/mL) added to the plates.
+
Both plates were left to incubate longer as the slow growth may be due to the high concentration of Kanamycin (50mg/mL) added to the plates.
==8/26/2010==
==8/26/2010==
Line 594: Line 596:
Attempted to perform electroporation according to [[Media:Electroporation Protocol 2.pdf|Electroporation Protocol 2]] with parts:
Attempted to perform electroporation according to [[Media:Electroporation Protocol 2.pdf|Electroporation Protocol 2]] with parts:
-
*pSB1AT3- Amp/Tet high copy backbone with RFP expression insert
+
*pSB1AT3 - Amp/Tet high copy backbone with RFP expression insert
-
*BBa_K145279- Tet resistance and Tet-activated GFP insert on Amp backbone
+
*BBa_K145279 - Tet resistance and Tet-activated GFP insert on Amp backbone
-
*BBa_I13504- Promoterless RBS-GFP insert on Amp backbone
+
*BBa_I13504 - Promoterless RBS-GFP insert on Amp backbone
-
*B0034- RBS on Amp backbone
+
*B0034 - RBS on Amp backbone
However, cells took too long to grow out because only 1 mL of overnight was used to inoculate the 100 mL fresh culture. OD600 was only 0.142 after 4.5 hours.
However, cells took too long to grow out because only 1 mL of overnight was used to inoculate the 100 mL fresh culture. OD600 was only 0.142 after 4.5 hours.
Line 609: Line 611:
The first set of overnight cultures are experiencing slow growth.  
The first set of overnight cultures are experiencing slow growth.  
-
A second set of overnight cultures were started in lower Kanamycin levels (5 mL LB + KAN 25 + 1 mM IPTG).
+
A second set of overnight cultures were started with lower Kanamycin levels (5 mL LB + KAN 25 + 1 mM IPTG).
==8/29/2010==
==8/29/2010==
Line 625: Line 627:
'''Digest of Flu + Kan Backbone Ligation'''
'''Digest of Flu + Kan Backbone Ligation'''
-
The miniprepped cells (from 8/29/2010) were digested with EcoRI & Spel. (To screen if the ligation worked properly or if mixed sites formed). 20 uL of DNA from ligation #1 and 25 uL of DNA from ligation #2 were used for the digest (to compensate for low miniprep yields).  
+
The miniprepped cells (from 8/29/2010) were digested with EcoRI & Spel to screen if the ligation worked properly or if mixed sites formed. 20 uL of DNA from ligation #1 and 25 uL of DNA from ligation #2 were used for the digest to compensate for the low miniprep yields.  
Line 634: Line 636:
Parts:
Parts:
-
*pSB1AT3 (13A)- Amp/Tet high copy backbone with RFP expression insert  
+
*pSB1AT3 (13A) - Amp/Tet high copy backbone with RFP expression insert
-
*BBa_K145279 (4L)- Tet resistance and Tet-activated GFP insert on Amp backbone
+
*BBa_K145279 (4L) - Tet resistance and Tet-activated GFP insert on Amp backbone
-
*BBa_I13504 (22L)- Promoterless RBS-GFP insert on Amp backbone
+
*BBa_I13504 (22L) - Promoterless RBS-GFP insert on Amp backbone
-
*B0034 (2M)- RBS on Amp backbone
+
*B0034 (2M) - RBS on Amp backbone
-
*O/N- 7:20 pm night before
+
*O/N - 7:20 pm night before
-
*Fresh culture- 11:45 am, grown out in Lin lab 30 degree shaker
+
*Fresh culture - 11:45 am, grown out in Lin lab 30 degree shaker
*Cells harvested at 3 pm with OD of 0.419
*Cells harvested at 3 pm with OD of 0.419
*1:100 dilution after wash step showed OD of 1.2
*1:100 dilution after wash step showed OD of 1.2
-
*Recovery was at 37 degrees for 1 hour
+
*Recovery was at 37°C for 1 hour
*Undiluted, 1:10, and 1:100 concentrations were plated for each sample
*Undiluted, 1:10, and 1:100 concentrations were plated for each sample
-
We didn't quite have enough comp cells, because we forgot to account for controls when making the cells, so a couple samples were a bit short (22L and another).
+
We didn't quite have enough comp cells because we forgot to account for controls when making the cells. A couple samples were a bit short (22L and another).
==8/31/2010==
==8/31/2010==
Line 652: Line 654:
'''Gel of Flu + Kan Backbone Ligation'''
'''Gel of Flu + Kan Backbone Ligation'''
-
A Gel was ran with the two digested ligations (from 8/30/2010) and failed to illuminate under UV light because of a mistake in the EtBr concentration used (2 mg/mL was used instead of the standard 10 mg/mL).
+
A Gel was run with the two digested ligations (from 8/30/2010) and failed to illuminate under UV light because of a mistake in the EtBr concentration used (2 mg/mL was used instead of the standard 10 mg/mL).
-
''' Miniprep of Flu Ligation #1 & #2 (2nd Set) and 2M'''
+
'''Miniprep of Flu Ligation #1 & #2 (2nd Set) and 2M'''
The Nanodrop values are as follows:
The Nanodrop values are as follows:
Line 684: Line 686:
'''Overnight cultures of Flu Ligation 1 & 2 (set 3), KAN BB, 2M'''
'''Overnight cultures of Flu Ligation 1 & 2 (set 3), KAN BB, 2M'''
-
The Flu ligations and KAN Backbone cultures were started in 5 mL of LB + KAN 25 + 1 mM IPTG
+
The Flu ligations and KAN Backbone cultures were started in 5 mL of LB + KAN 25 + 1 mM IPTG.
-
The 2M culture was started in 5 mL of LB + AMP
+
The 2M culture was started in 5 mL of LB + AMP.
==9/4/2010==
==9/4/2010==
Line 714: Line 716:
Made dilution protocol for CHCA starting with 1500 mg/L stock solution made by Ann previously. Made 500, 250, 125, 50, 10, 5, and 1 mg/L solutions, with at least 0.75 mL left of each after dilutions. These were stored in the fume hood and then given to Mike for analysis on the HPLC.
Made dilution protocol for CHCA starting with 1500 mg/L stock solution made by Ann previously. Made 500, 250, 125, 50, 10, 5, and 1 mg/L solutions, with at least 0.75 mL left of each after dilutions. These were stored in the fume hood and then given to Mike for analysis on the HPLC.
-
Also, autoclaved liquid waste.
+
Also, liquid waste was autoclaved.
==9/12/2010==
==9/12/2010==
Line 777: Line 779:
[[Image:28 Flu PCR Gel.jpg|400px]]
[[Image:28 Flu PCR Gel.jpg|400px]]
-
It appears samples 3 and 4 were successful, however the gel needs to be re-run because of excessive smearing. Jeremy Minty suggested lower voltage and less time. Also, "nonspecific" bands seen by Ann and Bryce were nearly nonexistent and also seen in lanes 1 and 2, suggesting that they are not nonspecific product, but something else, such as primer-dimer.
+
It appears samples 3 and 4 were successful. However, the gel needs to be re-run because of excessive smearing. Jeremy Minty suggested lower voltage and less time to remedy this. Also, "nonspecific" bands seen by Ann and Bryce were nearly nonexistent and also seen in lanes 1 and 2, suggesting that they are not nonspecific product, but something else, such as primer-dimer.
==9/29/2010==
==9/29/2010==
Line 792: Line 794:
*KT2440 inoculated into 2 mL LB in 15mL tube
*KT2440 inoculated into 2 mL LB in 15mL tube
*others inoculated into 2 mL LB + 100μg/mL amp in 15mL tube
*others inoculated into 2 mL LB + 100μg/mL amp in 15mL tube
-
*all incubated in 30°C, 200rpm shaking -- ERB 1230, 8:45pm
+
*all incubated in 30°C, 200rpm shaking - ERB 1230, 8:45pm
Streaked KT2440 on LB plate from frozen
Streaked KT2440 on LB plate from frozen
-
*incubated 37°C -- ERB 1239
+
*incubated 37°C - ERB 1239
Added KT2440 to strain database
Added KT2440 to strain database
Mixed M9 A and B solutions and diluted 1/50 in 200 mL sterile DIwater -- in 4 50mL tubes
Mixed M9 A and B solutions and diluted 1/50 in 200 mL sterile DIwater -- in 4 50mL tubes
Line 814: Line 816:
Repeated Flu PCR Gel from yesterday, with the following variations:
Repeated Flu PCR Gel from yesterday, with the following variations:
*Regular amount of product/loading dye (4.8 and 1.2 uL)
*Regular amount of product/loading dye (4.8 and 1.2 uL)
-
*Ran at 86V for 60 min
+
*Run at 86 V for 60 min
[[Image:29 Flu PCR Gel.jpg|400px]]
[[Image:29 Flu PCR Gel.jpg|400px]]
Line 824: Line 826:
Aliquot M9 and buffered M9 into five 15mL tubes each
Aliquot M9 and buffered M9 into five 15mL tubes each
 +
Need 15 mL stocks of each of the following media -- each 15 mL neutral and 15 mL buffered at pH 9:
Need 15 mL stocks of each of the following media -- each 15 mL neutral and 15 mL buffered at pH 9:
*M9
*M9
Line 832: Line 835:
*LB
*LB
Added 150 μL 40% glucose to appropriate tubes (.4% final concentration)
Added 150 μL 40% glucose to appropriate tubes (.4% final concentration)
 +
Aliquot 15 mL LB into each of two 15mL tubes
Aliquot 15 mL LB into each of two 15mL tubes
 +
Added NAs to appropriate tubes
Added NAs to appropriate tubes
  - Acros: (.25 mg/mL)*(15mL)/(.91 mg/μL) = '''4.12 μL'''
  - Acros: (.25 mg/mL)*(15mL)/(.91 mg/μL) = '''4.12 μL'''
  - Sigma: (.25 mg/mL)*(15mL)/(.92 mg/μL) = '''4.08 μL'''
  - Sigma: (.25 mg/mL)*(15mL)/(.92 mg/μL) = '''4.08 μL'''
*added 4.1 μL of each NA to each respective tube
*added 4.1 μL of each NA to each respective tube
-
Added borax to one tube of LB to buffer at pH9
+
Borax was added to one tube of LB to buffer at pH9:
  - (190.7 mg)*(15μL/50μL) = '''57.2 mg''' per 15mL LB
  - (190.7 mg)*(15μL/50μL) = '''57.2 mg''' per 15mL LB
*tested pH with indicator strip
*tested pH with indicator strip
Line 863: Line 868:
''Marcus''
''Marcus''
-
Purified Flu PCR product with Qiagen PCR purification kit, and eluted in ultra pure H2O instead of EB. 35 uL remaining from samples 3 and 4 of PCR were combined and purified, and stored at 4 C.
+
Purified Flu PCR product with Qiagen PCR purification kit and eluted in ultra pure H2O instead of EB. 35 uL remaining from samples 3 and 4 of PCR were combined and purified, and stored at 4 C.
Bryce came in in the evening and got the concentration of the purified samples, along with some minipreps according to the [[Media:Nanodrop_Protocol.pdf|Nanodrop]] protocol:
Bryce came in in the evening and got the concentration of the purified samples, along with some minipreps according to the [[Media:Nanodrop_Protocol.pdf|Nanodrop]] protocol:
-
Flu PCR Product- 29.8 ng/uL
+
Flu PCR Product - 29.8 ng/uL
-
13A (pSB1AT3)- 240.5 ng/uL
+
13A (pSB1AT3) - 240.5 ng/uL
-
22L (BBa_I13504)- 467.4 ng/uL
+
22L (BBa_I13504) - 467.4 ng/uL
-
2M (B0034)- 661.4 ng/uL
+
2M (B0034) - 661.4 ng/uL
Bryce also digested the Flu operon and pSB1AT3 with EcoRI and SpeI according to the [[Media:General_DNA_Digest_Protocol.pdf|DNA Digest]] protocol. He used 17 uL of the Flu PCR product and 5 uL of pSB1AT3.
Bryce also digested the Flu operon and pSB1AT3 with EcoRI and SpeI according to the [[Media:General_DNA_Digest_Protocol.pdf|DNA Digest]] protocol. He used 17 uL of the Flu PCR product and 5 uL of pSB1AT3.

Latest revision as of 01:36, 27 October 2010


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Back to Oil Sands Main Notebook

8/1/2010

Nanodrop of pBAD and GFP verification

This miniprep was performed for the two cultures started last night according the the MODIFIED miniprep protocol. A frozen stock was made of the pBAD biobrick and placed in the -80°C freezer.

The DNA concentrations reported by the nanodrop are as follows:

  • pBAD: 386 ng/uL (260/280=2.02 and 260/230=2.32)
  • GFP: 25.8 ng/uL (260/280=2.22 and 260/230=2.72)

The pBAD grew out very densely; when IPTG is added, the copy number is over 100. The DNA concentration was very high.

Digest of pBAD and GFP verification

The parts were digested according to the protocol in the protocol section of the wiki for a 50 uL reaction volume:

  • GFP #3 was digested with XbaI and PstI
  • GFP #4 was digested with EcoRI and XbaI
  • pBAD #1 was digested with EcoRI and SpeI
  • pBAD #2 was digested with SpeI and PstI

8/2/2010

Gel of Digested Parts

8-2-2010 gel of pbad and gfp annotated.jpg

  • Lane 1: Invitrogen 1 kb Plus ladder
  • Lane 2: GFP #3 cut with XbaI and PstI (GFP: 720 bp; Backbone: 2079 bp)
  • Lane 3: GFP #4 cut with EcoRI and XbaI
  • Lane 4: uncut GFP plamid (only 1 uL left so did not show up on gel)
  • Lane 5: pBAD cut with EcoRI and SpeI (pBAD: 1210 bp; Backbone: 4425 bp)
  • Lane 6: pBAD cut with SpeI and PstI
  • Lane 7: uncut pBAD (loaded very small amount into gel by accident)
  • Lane 8: uncut pBAD (loaded 1 uL of miniprep into gel)

We did not get the pBAD part due to the miniprep culture overgrowing and the cells lysing their chromosomal DNA (resulting in extremely long bands in the gel). We will try miniprepping the DNA from this culture again letting the culture grow out for a shorter amount of time before the miniprep.

flu primers

Primers were ordered today to amplify the flu operon of E. coli K12 by colony PCR:

8/3/2010

Minimal Media

Minimal media was mixed today according to the following recipes (also found in the media recipe section of the notebooks):

Culturing Bacteria in Naphthenic Acids (NAs)

The following 9 cultures were started to test bacteria growth in NA media:

  • Negative Control (to make sure all strains grow in this minimal media)
    • E. coli K12 in Bushnell-Haas minimal media supplemented with glucose
    • P. putida oilsands in Bushnell-Haas minimal media supplemented with glucose
    • P. flourescens oilsands in Bushnell-Haas minimal media supplemented with glucose
  • NA media
    • E. coli K12 in Bushnell-Haas minimal media supplemented with cyclohexane carboxylic acid (120 mg/L concentration)
    • P. putida oilsands in Bushnell-Haas minimal media supplemented with cyclohexane carboxylic acid (120 mg/L concentration)
    • P. flourescens oilsands in Bushnell-Haas minimal media supplemented with cyclohexane carboxylic acid (120 mg/L concentration)
  • pH adjusted NA media
    • E. coli K12 in Bushnell-Haas minimal media supplemented with cyclohexane carboxylic acid (120 mg/L concentration), pH adjusted between 8 and 9
    • P. putida oilsands in Bushnell-Haas minimal media supplemented with cyclohexane carboxylic acid (120 mg/L concentration), pH adjusted between 8 and 9
    • P. flourescens oilsands in Bushnell-Haas media supplemented with cyclohexane carboxylic acid (120 mg/L concentration), pH adjusted between 8 and 9

1 mL of the above media was added to a 15 mL falcon tube and inoculated with -80°C freezer stock of each culture and grown in the 30C shaker (Psuedomonas strains are temperature sensitive). The cultures were started at 7:30 pm.

8/4/2010

Culturing Bacteria in Naphthenic Acids (NAs)

At 12:30 pm the OD600 of the cultures started yesterday were measured and are listed below. These results must be taken with a grain of salt, because the cultures were started from the -80°C freezer stock with different amounts of inoculum:

  • P. putida BH-glu: 0.244
  • P. putida BH-CHCA: 0.0245
  • P. putida BH-CHCA pH 9: 0.033
  • P. flourescens BH-glu: 0.396
  • P. flourescens BH-CHCA: 0.219
  • P. flourescens BH-CHCA pH9: 0.417
  • E. coli K12 BH-glu: 0.017
  • E. coli K12 BH-CHCA: 0.016
  • E. coli K12 BH-CHCA pH9: 0.0325

P. flourescens oilsands seems to love this medium and grew out very densely! P. putida (oilsands) showed signs of growing. E. coli K12 did not grow at all and pellets of dead cells appeared on the bottom of the culture flask.

From these results we can start cultures for the biofilm assay experiment and time the cultures so they all become dense around the same day. If all cultures are started 24 hours in advance they all should saturate by the next day.

The E. coli K12 and P. putida oilsands cultures were placed back in the 30°C shaker to keep growing. By 9:30pm, the P. putida oilsands cultures had saturated and the E. coli K12 cultures had still not grown.

As a control for the biofilm assay E. coli K12 will be grown in M9 minimal media with glucose as a carbon source with and without casamino acids. We will not attempt to grow E. coli K12 in Bushnell-Haas media.


Measuring pH of Bushnell-Haas minimal media with cyclohexane carboxylic acid adjusted to pH 9

Today Marc showed us how to use the pH meter in the Lin Lab. Yesterday the pH of the media was estimated by pH paper to be between 8 and 9. The actual pH of the media is 7.35. The pH of the already adjusted media from yesterday was increased by added more 250 uL of 0.1 M NaOH to 5mL of BH-CHCA media and the pH read 7.6 using the pH probe. Another 250 uL of NaOH was added to this mixture and the pH read 8.8 using the pH probe. pH paper was dotted using this mixture to see the exact color it should turn when the pH is close to 9 (the paper looks more blue than green when the dot first appears).

A more concentrated NaOH sterilized stock solution needs to be made so when NaOH is added to the media the salts are not diluted


Bacterial Growth in pH 9

After determining that 1 mL of the supposedly already adjusted BH-CHCA media needed 100 uL of 0.1 M NaOH solution per mL to really be close to a pH of 9, new overnight cultures were started of the two Pseudomonas oilsands strains in 1 mL of the BH-CHCA pH-adjusted media from yesterday with an additional 100 uL of 0.1 M NaOH added. These two cultures were placed in the 30°C shaker to grow out overnight at 8:30 pm.


Miniprep of pBAD, take 2

At 10:45 pm a 5 mL culture of the pBAD biobrick was started from the -80°C freezer stock in LB + 25 mg/mL KAN + 1 mM IPTG for a miniprep tomorrow morning.

PCR of flu operon of E. coli K12

The primers ordered on Monday came in today!

PCR reagents were picked up from the life science store.

  • dNTPs
    • These dNTPs came in four separate containers individually in a 100 mM solution. The following recipe was used to make the 10 mM dNTP mixture:
      • 100 uL of 100 mM dATP
      • 100 uL of 100 mM dTTP
      • 100 uL of 100 mM dGTP
      • 100 uL of 100 mM dCTP
      • 600 uL of ultra pure water
  • Phusion High Fidelity Polymerase:
    • comes with buffer
    • used to amplify long pieces of DNA
  • 50 rxn PCR purification kit

The PCR was run according to the following colony PCR protocol

The final annealing cycle for 10 minutes at 72°C was accidentally added in the last 30 cycle loop. This mistake was caught after 3 cycles and the PCR was stopped, the program fixed and the reaction restarted.

Gel of flu colony PCR

The following gel was run in a 0.7% agarose gel at 85 V according to the protocol on the wiki:

8-4-2010 PCR of flu operon.jpg

For the first 5 cycles the annealing temperature was lowered to the Tm of the 16 or 18 bp that was complementary to the E. coli K12 template. A gradient of temperature was run for the annealing temperatures of the first five cycles as labeled in the gel.

8/5/2010

Bacterial Growth at pH 9

At 9:00am the two cultures started in the BH-CHCA were tested to ensure the pH was around 9. Both P. putida oilsands and P. fluorescens oilsands showed slight signs of growth. Later, in the day at 3:30pm, the cultures were denser; the OD600 of the culture was:

  • P. putida oilsands: 0.065
  • P. fluorescens oilsands: 0.433

It should be noted some of the salts precipitated out of solution in the 1 mL cultures.

Miniprep of pBAD, take 2

The miniprep was started at 8:45am. After 10 hours of growth the culture was saturated and the miniprep initialized according to the modified miniprep protocol.

Nanodrop of pBAD, take 2

The concentration of the pBAD promoter miniprepped earlier today is 418 ng/uL. It is most likely still contaminated with this high concentration but we will still digest it and run a gel to check.

Digest of pBAD, take 2

This digest was performed according to the digest protocol in the protocol section of the wiki:

pBAD #3 was digested with EcoRI and SpeI pBAD #4 was digested with SpeI and PstI

PCR of flu operon

The PCR of the flu operon was repeated 3 more times at higher annealing temperatures as this showed to have less unspecific product (shown by the gel from yesterday) according to this: modified colony PCR protocol. The volumes of the reactions were increased to 50 uL for a PCR purification step afterwards.

Gel of flu operon PCR and pBAD promoter digest

The gel ran exactly the same for the pBAD promoter as on 8/2/2010. The sample is probably contaminated and a new colony should be picked from the transformation plate and screened.

Also, no bands appeared for the flu PCR. One error might be because we did not include an initial cell lysis step. The PCR will be attempted again tomorrow.

Biofilm assay in minimal media

Cultures were started according to the biofilm assay protocol for the inoculation of the microplate; these will be used in the biofilm assay tomorrow.

8/6/2010

PCR of flu take 3

The first PCR of the flu operon was repeated with an added cell lysis step according to the following modified protocol:

Gel of PCR of flu take 3

8-6-2010 PCR of flu operon.jpg

Invitrogen 1 kb plus ladder

Due to the unspecific product the flu operon will be purified with gel purification.

Biofilm Assay

The OD600 of the cultures started yesterday are listed below along with the volume for resuspension after 1 mL of culture was pelleted:

OD600 Volume for resuspension (uL)
E. coli K12 LB 0.914 228
P. putida KT2440 LB 1.168 292
P. putida oilsands LB 1.119 280
P. fluorescens oilsands LB 1.283 321
E. coli K12 M9 minimal media plus glucose 0.391 98
P. putida KT2440 Bushnell-Hass media plus glucose 0.545 136
P. putida oilsands Bushnell-Hass media plus glucose 0.385 96
P. fluorescens oilsands Bushnell-Hass media plus glucose 0.504 126
E. coli K12 M9 minimal media plus glucose plus casamino acids 0.814 204
P. putida KT2440 Bushnell-Hass media plus glucose plus casamino acids 1.185 296
P. putida oilsands Bushnell-Hass media plus glucose plus casamino acids 1.121 280
P. fluorescens oilsands Bushnell-Hass media plus glucose plus casamino acids 1.191 298
P. putida KT2440 Bushnell-Hass media plus cyclohexane carboxylic acid 0.0345 8.6
P. putida oilsands Bushnell-Hass media plus cyclohexane carboxylic acid 0.144 36
P. fluorescens oilsands Bushnell-Hass media plus cyclohexane carboxylic acid 0.197 49
P. putida KT2440 Bushnell-Hass media plus cyclohexane carboxylic acid pH9 0.034 8.5
P. putida oilsands Bushnell-Hass media plus cyclohexane carboxylic acid pH9 0.002 0.06
P. fluorescens oilsands Bushnell-Hass media plus cyclohexane carboxylic acid pH9 0.199 49.8

Not all of the cultures grew out (P. putida KT2440 Bushnell-Haas media plus cyclohexane carboylic acid, P. putida KT2440 Bushnell-Haas media plus cyclohexane carboylic acid pH 9, P. putida oilsands Bushnell-Haas media plus cyclohexane carboxylic acid pH 9). The modified bio assay protocol was used to inoculate the microplate.

The microplate was inoculated at 4:30pm.

8/8/2010

Biofilm Assay

The washing and CV staining of the bioassay was performed according to the modified protocol on 8/6/2010.


The OD 600 of the plate after the bioassay growth is shown in the following graph:

8-8-2010 OD 600 after biofilm growth graph.jpg


The Crystal Violet OD 600 after staining is show in the following graph:

8-8-2010 CV OD 600 biofilm after staining.jpg


The Crystal Violet OD 600 to OD 600 after growth ratio is shown in the following graph:

8-8-2010 CV OD 600 to OD 600 ratio graph.jpg


The negative control (E. coli K12 in M9 minimal media with glucose) grew one of the best biofilms; this contradicted what was expected. From here we need to find a knock out strain that is deficient in forming biofilms.

A strong conclusion cannot be drawn about the biofilm forming abilities in minimal media with cyclohexane carboxylic acids (CHCA) as the cells did not grow very densely. This is the reason for the large error bars.

It is interesting that P. fluorescens in pH 9 did not form biofilms, even though the cell culture grew relatively well. We need a negative control to confirm this result.

8/9/2010

PCR of flu for gel extraction

Because there are unspecific products from the PCR of flu, a gel extraction will have to be used to isolate the desired DNA.

50 uL reaction volume for PCR was performed using 65.5C for the initial annealing temperature as shown in the following colony PCR protocol for flu. The higher annealing temperature reduces unspecific products.

Gel Extraction of flu operon

A gel extraction was performed according to the following gel extraction protocol.

20 uL of the flu #14 PCR was mixed with 5 uL of blue juice to run the gel.

The weight of the eppendorf tube was 1.0284 g.

The weight of the eppendorf tube with the sample was 1.2030 g.

The weight of the gel sample was 0.175 g.

524 uL of QG buffer was added to dissolve the gel sample.

No DNA appeared on the nanodrop. Marc ran the flu gel purified sample on his gel to confirm no DNA eluted.

DNA did appear on the gel that Marc ran so we do have DNA from the gel extraction. The nanodrop may not have been able to pick it up because it was too low of a concentration or it was contaminated (very high 260/230 reading).

8-9-2010 Gel extraction of flu.jpg

8/10/2010

Ann and Bryce

Transformation of Constitutive Promoter and KAN backbone

The transformation was performed according to the electroporation protocol in the protocol section of the wiki.

The comp cell culture was started at 9:10am by adding the 8 mL overnight culture into 40mL of LB media in a 500 mL sterile flask.

The OD600 of the culture was checked at 11:20am and was 1.0. This culture was slightly overgrown but was immediately placed on ice and used anyways (this might lower the efficiency of the procedure).

The biobricks we are transforming today are:

  • J23100 (constitutive promoter)
  • I14018 (PCR purify to remove promoter and be left with backbone that has KAN resistance)
  • (pBAD biobrick taken from the registry as a positive control)

The transformation was done at 2:15pm and the cells were plated on the following plates:

  • negative control (comp cells only) on LB+100 mg/mL AMP
  • constitutive promoter on LB+100 mg/mL AMP
  • negative control (comp cells only) on LB+25 mg/mL KAN + 1mM IPTG
  • positive control (pBAD promoter taken from the registry) on LB+25 mg/mL KAN + 1mM IPTG
  • backbone on LB+25 mg/mL KAN + 1mM IPTG

The plates were placed in the 37°C incubator to grow out overnight.

We plan on ligating the flu operon into the KAN backbone than cutting than inserting the promoter in front of this part. We must take this approach because the flu operon cannot be digested with PstI.


Marcus

Brought over 5 mL of each standard pH buffer (4, 7, 10) from Lin lab.

Started NA extraction from oil sand ore:

  • Made 1M NaOH solution in Lin lab
    • 1.969 g NaOH pellets
    • 50 mL dH2O
  • Mixed ore with 1M NaOH in 600 mL beaker
    • 51.3 g 1M NaOH
    • 38.6 g ore
  • Put beaker in shaker for 48 hours

See the NA Extraction Protocol.

8/11/2010

Miniprep of constitutive promoter and backbone with KAN resistance

The transformation was successful; both plates with the constitutive promoter and the backbone with KAN resistance grew up (even though the positive control did not grow...).

The constitutive promoter part is followed by RFP. We saw this on the transformation plates because the colonies were red.

A colony was picked from each plate and at 9:00am and 5mL cultures were started in the following media:

  • backbone: LB + 50 mg/mL KAN + 1mM IPTG
  • constitutive promoter: LB + 100 mg/mL AMP

These cultures were placed in the 37°C incubator to grow up.

At 9:00pm, the cultures were miniprepped according to the modified miniprep protocol.

Nanodrop of constitutive promoter and KAN backbone

The concentration of the minipreps are as follows:

  • backbone: 60 ng/uL (260/280= 1.87, 260/230= 2.06)
  • constitutive promoter: 200ng/uL (260/280= 1.89, 260/230= 2.30)

PCR of flu operon from the gel extraction product

Using gel extraction product for ligations lowers the efficiency of the ligation. To get around this problem we performed another PCR reaction using the gel extraction product as the template according to the following PCR protocol.

Gel of PCR of flu operon from gel extraction product

There was more unspecific product from trying the PCR this reaction from the gel extracted part, so the gel extracted part will be used for further genetic manipulations.

8/12/2010

Digest of constitutive promoter, KAN backbone and flu operon

The following digests were performed:

  • constitutive promoter: EcoRI and SpeI (labeled P ES)
  • constitutive promoter: EcoRI and XbaI (labeled P EX)
  • KAN backbone: EcoRI and SpeI (labeled B ES)
  • KAN backbone: SpeI and PstI (labeled B SP)
  • flu operon (from gel purified product): EcoRI and SpeI (labeled F ES)

All digests except for the KAN backbone digested with EcoRI and SpeI were 20 uL volumes. 1 uL of each enzyme was added along with 0.5 uL of BSA to the 20 uL reactions and the rest of the reagents were ajusted by multiplying the 50 uL volume by 0.4. To make up for the extra volume of BSA and enzymes, the extra amount was subtracted from the amount of water added.

Because the KAN backbone digested with EcoRI and SpeI is going to be PCR purified, double the amount of DNA was added and a 50 uL volume reaction was performed.

Since no nanodrop reading was made for the flu operon, no water was added and only the gel extraction was used.

Gel of constitutive promoter, KAN backbone and flu operon

8-13-2010 get of flu KAN ligation.JPG

All parts digested accordingly and are ready for the ligation.

PCR purification of KAN backbone

The PCR purification was performed with the 50 uL digest of the KAN backbone with EcoRI and SpeI according to the protocol in the protocol section of the wiki.

250 uL of buffer PB was used to mix with the digest before adding the mixture to the column.

Nanodrop of PCR purified KAN backbone

The PCR purified KAN backbone had a concentration of 14.8 ng/uL with a 260/280 ratio of 1.86 and a 260/230 ratio of 0.91. These ratios indicate that the sample is contaminated but there is still a high enough peak at 260 to show DNA is present. Another gel could be run to verify this, but this step will be skipped and the DNA will be used for the ligation.

Ligation of flu operon with the KAN backbone

The ligation was performed according to the following T4 ligase protocol.


Marcus

Removed ore/NaOH extraction mixture from shaker. Poured liquid into 50 mL tube, and strained the solids through a coffee filter and funnel to collect as much liquid as possible; 20mL was recovered. This was filtered through a 50 mL Steriflip vacuum tube; however, the filter became clogged and a second one was used. This required an extended vacuum time (in the future the mixture should be centrifuged first for several minutes). Recovered a total of about 40 mL (???).

8/13/2010

Marcus

  • Autoclaved 1.5 mL tubes.
  • Took two bags of autoclave waste to DOW building to be autoclaved.

8/14/2010

Transformation of the Flu Operon + Kan backbone ligation

The Heat shock transformation protocol was used.

E.Coli DH5α was used as competent cells and had a prewashing OD600 of .845 and a post washing OD600 of .787.

Two separate transformations were performed both with 100 uL Comp. Cells + 10 uL Flu + KAN BB Ligation.

Both transformations were plated on LB+Kan+IPTG(30 uL) and are currently incubating at 37 C in the ERB.

8/16/2010

Transformation of Flu Operon + Kanamycin Backbone Ligation

  • The first transformation plate showed no colonies.
  • The second transformation plate showed minimal colonies.

Both plates were left to incubate longer as the slow growth may be due to the high concentration of Kanamycin (50mg/mL) added to the plates.

8/26/2010

Overnight Cultures of the Flu Operon + Kanamycin Backbone Transformation

Colonies appeared on both transformation plates.

Overnight cultures were started from both plates in 5 mL LB + KAN 50 + 1 mM IPTG.


Marcus

Attempted to perform electroporation according to Electroporation Protocol 2 with parts:

  • pSB1AT3 - Amp/Tet high copy backbone with RFP expression insert
  • BBa_K145279 - Tet resistance and Tet-activated GFP insert on Amp backbone
  • BBa_I13504 - Promoterless RBS-GFP insert on Amp backbone
  • B0034 - RBS on Amp backbone

However, cells took too long to grow out because only 1 mL of overnight was used to inoculate the 100 mL fresh culture. OD600 was only 0.142 after 4.5 hours.

Made 10 Amp and 10 LB plates.

8/27/2010

Overnight Cultures of the Flu Operon + Kanamycin Backbone Transformation Take 2

The first set of overnight cultures are experiencing slow growth.

A second set of overnight cultures were started with lower Kanamycin levels (5 mL LB + KAN 25 + 1 mM IPTG).

8/29/2010

Miniprep & Nanodrop of Flu + Kan Backbone Ligation

Before the miniprep .5 mL of both cultures were used to make a frozen stock. The frozen stocks now reside in the -20 C freezer in the ERB.

The first set of overnight cultures were miniprepped and eluted in EB Buffer.

After Nanodropping the DNA Concentrations for plated #1 and #2 were 9.5 and 4.0 ng/uL, respectively.

8/30/2010

Digest of Flu + Kan Backbone Ligation

The miniprepped cells (from 8/29/2010) were digested with EcoRI & Spel to screen if the ligation worked properly or if mixed sites formed. 20 uL of DNA from ligation #1 and 25 uL of DNA from ligation #2 were used for the digest to compensate for the low miniprep yields.



Marcus and John

Repeated electroporation attempted on 8/26 using Electroporation Protocol 2.

Parts:

  • pSB1AT3 (13A) - Amp/Tet high copy backbone with RFP expression insert
  • BBa_K145279 (4L) - Tet resistance and Tet-activated GFP insert on Amp backbone
  • BBa_I13504 (22L) - Promoterless RBS-GFP insert on Amp backbone
  • B0034 (2M) - RBS on Amp backbone
  • O/N - 7:20 pm night before
  • Fresh culture - 11:45 am, grown out in Lin lab 30 degree shaker
  • Cells harvested at 3 pm with OD of 0.419
  • 1:100 dilution after wash step showed OD of 1.2
  • Recovery was at 37°C for 1 hour
  • Undiluted, 1:10, and 1:100 concentrations were plated for each sample

We didn't quite have enough comp cells because we forgot to account for controls when making the cells. A couple samples were a bit short (22L and another).

8/31/2010

Gel of Flu + Kan Backbone Ligation

A Gel was run with the two digested ligations (from 8/30/2010) and failed to illuminate under UV light because of a mistake in the EtBr concentration used (2 mg/mL was used instead of the standard 10 mg/mL).

Miniprep of Flu Ligation #1 & #2 (2nd Set) and 2M

The Nanodrop values are as follows:

  • Flu #1 - 23.2 ng/uL
  • Flu #2 - 18.8 ng/uL
  • 2M - 17.8 ng/uL

9/1/2010

Gel of Flu + Kan Backbone Ligation (Take 2)

A Gel was ran with the same two digested ligations (from 8/30/2010) and failed to illuminate under UV light. The source of error is believed to be that the DNA ran off the gel because the voltage was originally set at 85V (to run for 1.5 hrs) and upon returning (after 1.5 hrs) the voltage had been changed to 105V (by another lab member roughly 15 minutes into the start of the gel).

9/2/2010

Digest of Flu + Kan Backbone Ligation (Take 2)

The miniprepped cells (from 8/31/2010) were digested with Xba1 (To screen if the ligation worked by checking for a band at 7790 base pairs). Ann's digest calculator was used to calculate the proper amount of DNA to use.

Miniprep of 22L, 13A, and Kan Backbone

The DNA concentrations from the Miniprep by Nanodropping are as follows:

  • 22L - 183.7 ng/uL
  • 13A - 54.7 ng/uL
  • KAN BB - 188.1 ng/uL

9/3/2010

Overnight cultures of Flu Ligation 1 & 2 (set 3), KAN BB, 2M

The Flu ligations and KAN Backbone cultures were started in 5 mL of LB + KAN 25 + 1 mM IPTG.

The 2M culture was started in 5 mL of LB + AMP.

9/4/2010

Miniprep of of Flu Ligation 1 & 2 (set 3), KAN BB, 2M

The DNA concentrations from the Miniprep by Nanodropping are as follows:

  • Flu #1 - 24.1 ng/uL
  • Flu #2 - 22.8 ng/uL
  • 2M - 44.0 ng/uL
  • KAN BB - 24.4 ng/uL

9/5/2010

Gel of Flu Ligation Transformation (take 3)

Flu 9-5 Zoom.jpg



Marcus

Ore extracts were combined into a single tube and re-filtered, then stored in acid storage cabinet.

9/11/2010

Marcus and John

Made dilution protocol for CHCA starting with 1500 mg/L stock solution made by Ann previously. Made 500, 250, 125, 50, 10, 5, and 1 mg/L solutions, with at least 0.75 mL left of each after dilutions. These were stored in the fume hood and then given to Mike for analysis on the HPLC.

Also, liquid waste was autoclaved.

9/12/2010

Alex

Made E. coli K12 broth culture from plate

  • 2 mL LB in 15mL tube
  • 37°C, 200 rpm shaking - ERB 1230

Uploaded NanR cloning protocol

9/24/2010

Marcus, Bryce, and Ben

Ran PCR of Flu operon according to the NanR cloning PCR protocol, with modifications as follows:

  • 1x PCR Mix:
    • 36.25 uL Ultra pure H2O
    • 10 uL 5x Phusion Buffer
    • 1 uL 10 mM dNTPs
    • 0.625 uL Forward Flu Primer
    • 0.625 uL Reverse Flu Primer
    • 1 uL DNA Template (1:10 O/N dilution)
    • 0.5 uL Phusion polymerase
  • PCR Program (FLUPCR)
    • 1. 96 C for 6 min
    • 2. 98 C for 10 sec
    • 3. 65.5 C for 30 sec
    • 4. 72 C for 1 min 45 sec
    • 5. Return to step 2 four times
    • 6. 98 C for 10 sec
    • 7. 67 C for 30 sec
    • 8. 72 C for 1 min 45 sec
    • 9. Return to step six 29 times
    • 10. 72 C for 10 min
    • 11. 4 C store

Unfortunately, a math mistake was made while making the master mix, which was discovered due to having too much master mix left over. The volumes used to create the master mix were:

  • 181.25 uL Ultra pure H2O
  • 100 uL 5x Phusion Buffer
  • 10 uL 10 mM dNTPs
  • 6.25 uL Forward Flu Primer
  • 6.25 uL Reverse Flu Primer
  • 10 uL DNA Template

2.5 uL Phusion polymerase

9/27/2010

Marcus

Got some chloramphenicol from the Lin lab (34 mg/mL 1000x stock) and made 10 chloramphenicol plates and 10 LB plates. Also, made 45 mL of a LB+CM34 stock, and autoclaved 500 mL of LB.

9/28/2010

Marcus

Ran gel of Flu PCR product. Used the Gel Electrophoresis protocol with the following variations:

  • Doubled PCR product volume, in case gel extraction was needed
    • 9.6 uL PCR product, 2.4 uL Blue Juice
  • Ran at 110V for 50 min

28 Flu PCR Gel.jpg

It appears samples 3 and 4 were successful. However, the gel needs to be re-run because of excessive smearing. Jeremy Minty suggested lower voltage and less time to remedy this. Also, "nonspecific" bands seen by Ann and Bryce were nearly nonexistent and also seen in lanes 1 and 2, suggesting that they are not nonspecific product, but something else, such as primer-dimer.

9/29/2010

Alex and John

Dissolved M9 salts to 50X

  • All of component A in 1000 mL DIwater
  • All of component B in 1000 mL DIwater

Autoclaved both: each split into two 1000mL bottles filled w/ 500mL each (four bottles total)

  • 30 min sterilize time

Began biofilm assay following Alex's biofilm assay protocol

Made broth cultures of P. putida LD1, P. fluorescens LD2, and LD1+LD2 coculture from plates, and P. putida KT2440 from Lin Lab frozen stock

  • KT2440 inoculated into 2 mL LB in 15mL tube
  • others inoculated into 2 mL LB + 100μg/mL amp in 15mL tube
  • all incubated in 30°C, 200rpm shaking - ERB 1230, 8:45pm

Streaked KT2440 on LB plate from frozen

  • incubated 37°C - ERB 1239

Added KT2440 to strain database Mixed M9 A and B solutions and diluted 1/50 in 200 mL sterile DIwater -- in 4 50mL tubes

  • each tube 1 mL M9A, 1 mL M9B and 48 mL water

Alex

Uploaded Alex's biofilm assay protocol

Added borax to two of the M9 tubes (100 mL) to buffer at ~pH 9.2

- (.01 mol/L)*(.05 L)*(381.37 g/mol) = 190.7 mg borax/50 mL water
  • Tested pH with pH indicator strips
    • ~9
  • Vacuum-filtered 100 mL w/ steriflips

Marcus

Repeated Flu PCR Gel from yesterday, with the following variations:

  • Regular amount of product/loading dye (4.8 and 1.2 uL)
  • Run at 86 V for 60 min

29 Flu PCR Gel.jpg

Results look much better, bands are tighter, and conclusions are the same as yesterday. No gel extraction will be necessary.

9/30/2010

Alex

Aliquot M9 and buffered M9 into five 15mL tubes each

Need 15 mL stocks of each of the following media -- each 15 mL neutral and 15 mL buffered at pH 9:

  • M9
  • M9 + 0.4% glucose
  • M9 + Acros NAs (250mg/L)
  • M9 + Sigma Aldrich NAs (250mg/L)
  • M9 + both Acros & Sigma NAs (each 250mg/L)
  • LB

Added 150 μL 40% glucose to appropriate tubes (.4% final concentration)

Aliquot 15 mL LB into each of two 15mL tubes

Added NAs to appropriate tubes

- Acros: (.25 mg/mL)*(15mL)/(.91 mg/μL) = 4.12 μL
- Sigma: (.25 mg/mL)*(15mL)/(.92 mg/μL) = 4.08 μL
  • added 4.1 μL of each NA to each respective tube

Borax was added to one tube of LB to buffer at pH9:

- (190.7 mg)*(15μL/50μL) = 57.2 mg per 15mL LB
  • tested pH with indicator strip
    • ~9

Tested pH of neutral M9 with indicator strip

  • ~7

Moved KT2440 LB plate from 37°C to 4°C -- ERB 1239

Continued Alex's biofilm assay protocol

  • made one 96-well plate for all strains and media at pH7 only
  • plate template :

9-30 plate template.jpg

    • 100 μL/well: 99 μL medium + 1 μL culture
    • covered plate with gas-permeable membrane
  • Started 24-hr kinetic reading in Lin Lab microplate reader
    • 30°C
    • OD600 absorbance
    • read every 30 min

Made broth cultures for LD1, LD2 and KT2440 from yesterday's overnight broths

  • 2 mL each in a 15mL tube
  • 30°C, 200 rpm shaking - 6:20pm

Discarded yesterday's overnight broths


Marcus

Purified Flu PCR product with Qiagen PCR purification kit and eluted in ultra pure H2O instead of EB. 35 uL remaining from samples 3 and 4 of PCR were combined and purified, and stored at 4 C.

Bryce came in in the evening and got the concentration of the purified samples, along with some minipreps according to the Nanodrop protocol:

Flu PCR Product - 29.8 ng/uL 13A (pSB1AT3) - 240.5 ng/uL 22L (BBa_I13504) - 467.4 ng/uL 2M (B0034) - 661.4 ng/uL

Bryce also digested the Flu operon and pSB1AT3 with EcoRI and SpeI according to the DNA Digest protocol. He used 17 uL of the Flu PCR product and 5 uL of pSB1AT3.