Team:Michigan/Oil Sands

Oil Sands Lab Notebook

This team includes Ann Lesnefsky, Bryce Rajabian, Marcus Lehr, Alex Pyden, Jeremy Golden, John Seong Kyu Yang and Kilho Lee.

Results to Date

10/1/10 pH7 Biofilm Assay

10/1/10 pH7 Growth Curves

10/3/10 pH9 Growth Curves

10/3/10 pH9 Biofilm Assay

6/28/2010

Pseudomonas putida KT2440 Antibiotic Resistance

• Protocol for starting overnight culture from -80°C freezer
• All protocols can also be found in the Protocols section of the Notebook

6/29/2010

Pseudomonas putida KT2440 Antibiotic Resistance

• Protocol for making frozen stocks

6/29/2010 and 6/30/2010

Pseudomonas putida KT2440 Antibiotic Resistance

• Protocol for P. putida KT2440 antibotic resistance

7/1/2010

Literature Review Napthenic Acids Composition & HPLC Analysis

• Literature Review of the composition of napthenic acids in oil sands

7/2/2010

• Literature Review of HPLC columns for analysis of napthenic acids

Pseudomonas putida KT2440 Antibiotic Resistance

• Results

7/7/2010

Ann

Biobrick Transformation with Alex and Jennifer

• Made CaCl2 solution, and 100 mg/mL amphicilin stock solution according to the media section of the wiki
• Started an overnight culture of DH5alpha according to the heat shock transformation protocol
  • A 12 mL culture was started because we were multiplying the protocol by 4

7/8/2010

Ann

Biobrick Transformation with Marc, Katie and Audra according to the heat shock transformation protocol

• Started culture for biobrick transformation from over night at 2:30
• At 5:00pm the cultures had overgrown to an OD600 of 1.2
  • A a 1:3 dilution of cells was performed and the cells were allowed to go through another doubling period of 20 minutes
• The OD600 was measured again and found to be around 0.500
• the cultures were placed on ice at 5:30 and allowed to chill for 20 minutes
• The washings were performed and the comp cells were resuspended in the residual CaCl2 only
• The OD600 of the comp cells without the glycerol was above 0.3 therefore the comp cells were concentrated enough
• The heat shock was performed for the following biobricks
  • BBa_179015
  • BBa_179005
  • BBa_145001
  • BBa_117008
  • BBa_117002
  • BBa_103006
• The cultures were placed in the 30C shaker to grow up for an hour at 30C at 9:00pm
• The cultures were plated at 10:00pm

7/9/2010

Ann

Biobrick Transformation with Josh, Prae, Charlie according to the miniprep protocol

• After 9 hours of growing at 37C, the plates did not have any colonies on them
• After 22 hours of growth plates that had grown had clear colonies
  • The positive and negative controls grew out accordingly
  • Only BBa_179015 and BBa_179005 had colonies (both from plate 1 and none from plate 2...)
    • BBa_179015 had over 100 colonies
    • BBa_179005 had 2 colonies
• 5 mL overnight cultures were started with 100 mg/mL amp at 8pm from a single colony on each plate

7/10/2010

Ann

Biobrick Transformation with Marcus, Bryce, Kilho, Josh, Charlie, Jeremy

Miniprep

• Made frozen stocks from overnight culture of miniprep
• Performed miniprep on BBa_179015 and BBa_179005
• Measured DNA concentration with the Nanodrop
  • BBa_179015-11 ng/mL
  • BBa_179005-40 ng/mL

Digest

• Ran according to the digest protocol in the protocol section
  • Cut parts with EcoRI and PstI
• Digested at 37C for 30 minutes

Gel

• Ran according to the protocol in the protocol section
• Used 8 well comb instead of 13 well comb so the samples were too dilute too see

7/12/2010

Ann

Biobrick Transformation with Jeremy

Gel

• Reran gel from 7/10/2010
  • Lane 1-Invitrogen 1 kb plus ladder
  • Lane 2-Digested Bba_179005
    • Ran out of undigested Bba_179005
  • Lane 3-Digested Bba_179015
  • Lane 4-Uncut miniprep plasmid Bba_179015

We found that we successfully extracted BBa_179015, T7-GFP, because there were expected bands at 906 and 2079 as faintly seen in the gel picture above. No appeared for biobrick Bba_179005, T7 promoter, so this biobrick will be transformed again.

Biobrick Transformation take 2 with Jeremy

Autoclaved DI water for transformation and autoclaved sterile containers

• to autoclave sterile containers fill with DI water and decant the water before you start the culture in the container

7/13/2010

Biobrick Transformation take 2 with Jeremy, Marcus, Josh, Kevin, Audra, Katie

Ann

Performed according to the electroporation transformation

• Started the culture at 9:05am
• Removed the culture at 12:05pm with an OD600 of 0.809
• The OD600 of the comp cells were 1.2 after washing
• The time constant for all electroporation were between 5.6 and 5.8 for the following biobricks
  • Bba_K117008
  • Bba_K117008 #2 (from resuspending remaining part left in registry in 15 uL of ultra pure water)
  • Bba_K117002
  • Bba_K145001
  • Bba_K103006
  • Bba_I719005
• The cultures were allowed to grow in the incubator from 2:15-4:00pm
  • The cultures started to clump after growing this time
• 100 uL of cells were plated on 100 mg/mL AMP plates

7/14/2010

Biobrick Transformation take 2

Ann

Electroporation Transformation

All of the transformation plates grew out! (minus the negative control of course)

This means we should do all transformations by electroporation from now on. Just check with the Lin Lab to make sure we can use the electroporation machine for a few hours before starting

Miniprep

• Started overnight cultures in 5 mL of LB plus 100 mg/mL AMP
  • The following biobricks were started from the transformation plate
    • Bba_K117008
    • Bba_K117002
    • Bba_K145001
    • Bba_K103006
    • Bba_I719005
  • The following biobricks were started from the frozen stock in the -80C freezer
    • BBa_K719015

INPNC Biobrick part

• We received the INPNC biobrick (Bba_K265008) from UC Davis 2009 team. THANK YOU!!!
• It was shipped on LB plates in a pMA-SK plasmid from Mr. Gene in E. coli DH5alpha
• Since this plasmid has AMP antibiotic resistance I am pouring plates today with Marc and Kevin with 100 mg/mL AMP resistance to streak out the culture tomorrow with Josh

7/25/2010

Biobrick Transformation of suface display and pBAD

Ann

Tonight I started an 8mL culture of E. coli DH5alpha in LB broth and place in the 30C shaker to grow out overnight

7/26/2010

Biobrick Transformation of suface display and pBAD

Ann

Electroporation transformation

Started larger culture for comp cell preparation in 40mL of LB in a 500 mL sterile container at 9:00am

At 11:30am the OD of the culture was 0.730 and the cultures were placed on ice

The transformation of the biobricks was performed for according to the electroporation transformation protocol listed under the protocol section

The biobricks removed from the registry are as follows

• E0040 (GFP)
• K157013 (linker)
• I0500 (pBAD)

The miniprep of I79015 was used as a positive control and a negitive control was also run

The OD600 of the comp cells were not measured

All of the time constants for the transformation were above 5

the GFP, linker parts and the positive and negitive control were plated on 100 mg/mL AMP plates. The pBAD part and negitive control were plated on 50 mg/mL KAN

7/27/2010

Biobrick Transformation of suface display and pBAD

Transformation Results

The GFP and linker transformation worked and many colonies grew out on the 100 mg/mL AMP plates

The pBAD part did not grow out on the KAN plates. Upon further inspection this plasmid needs to be grown with IPTG to switch the origin of replication to a high copy above 100 copies per cell. Otherwise, another orgin of replication dominates that is less than 10 copies per cell and the antibiotic concentration of 50 mg/mL maybe too high. This transformation will have to be tried again

Miniprep of surface display

At 9:00am a 5 mL culture of the newly transformed GFP part, the newly transformed linker part, the ompA part transformed previously and the INP from the UC davis team were started for a miniprep in LB with 100 mg/mL AMP.

The modified miniprep protocol was used listed on the protocol section of the wiki to get a higher DNA concentration

At 9:00pm the GFP, linker and INP parts were miniprepped. The ompA culture had not grown out (b/c not enough -80C freezer stock was added) and a new culture was restarted at 11:00pm in 5 mL of LB with 100 mg/mL AMP.

Pouring Plates

More LB with 100 mg/mL AMP plates and LB with 25 mg/mL KAN plates were poured

7/28/2010

OmpA Miniprep

The OmpA culture grew out and was miniprepped according the the modified miniprep protocol at 11:30am

Nanodrop of surface display parts

The minipreped cultures from yesterday were nanodropped to determine the DNA concentration according to the protocol in the protocol section of the wiki

• GFP: 47.8 ng/uL
• linker: 33.2 ng/uL
• INP: 41.8 ng/uL
• OmpA: 49.3 ng/uL

Digest of surface display parts

The following biobricks were digested according to the protocol on the wiki with the enzymes listed below

• GFP 1: XbaI and PstI
• GFP 2: EcoRI and XbaI
• INP 1: EcoRI and SpeI
• INP 2: SpeI and PstI
• Linker 1: XbaI and PstI
• Linker 2: SpeI and PstI
• OmpA 1: EcoRI and SpeI
• OmpA 2: SpeI and PstI

Gel of Digest for surface display parts

A gel was run of the above digest with uncut plasmid as a control

• Lane 1: invitrogen 1 kb plus ladder
• Lane 2: GFP cut with XbaI and PstI (GFP: 720 bp Backbone: 2079 bp)
• Lane 3: GFP cut with EcoRI and XbaI
• Lane 4: uncut GFP plamid
• Lane 5:INP cut with EcoRI and SpeI (INP: 924 bp backbone: 2550 bp)
• Lane 6:INP cut with SpeI and PstI
• Lane 7:uncut INP plasmid
• Lane 8:Linker cut with XbaI and SpeI (Linker: 45 bp backbone: 2428 bp)
• Lane 9:Linker cut with SpeI and PstI
• Lane 10: uncut Linker plasmid
• Lane 11: OmpA cut with EcoRI and SpeI (OmpA: 464 bp backbone: 2079 bp)
• Lane 12: OmpA cut with SpeI and PstI
• Lane 13:uncut OmpA plasmid

7/29/2010

Biofilm Assay in LB media

Started cultures of E. coli K12, P. putida (oilsands) and P. fluorescens (oilsands) in 2 mL of LB and grew in the 30C shaker overnight

7/30/2010

Biofilm Assay in LB media

Today we carried out the following biofilm assay protocol

• @ 8:10am the cultures were started from the overnight
• @ 10:00am the OD600 of each culture was measured to be
  • E. coli K12: 0.8
  • P. fluorescens: 1.2
  • P. putida: 1.0
    • These cultures were overgrown but still used for the experiment

The results of this experiment are presented in the chart below

There is a large error in the P. putida strain because there was the largest variation in the OD 600 of the liquid culture which also resulted in a large variation in the CV OD 600 reading

We need to find a reference to compare our biofilms to. This could be either a strain that does not form biofilms or a strain that forms biofilms very well

Transformation of pBAD

Marcus and Alex tried the transformation of pBAD again according to the electroporation protocol with Ann's Notes

The recovery after electroporation was performed in 1 mL of LB with 1mM of IPTG added in order to have the pBAD part have a high copy number

30 uL of 1M IPTG solution was added to a 25 mg/mL KAN plate and spread with sterile beads in order to keep the high copy plasmid in the pBAD part while plating

7/30/2010

Transformation of pBAD

The transformation was successful and there were about 30 colonies on the plate. One colony was picked at 7:30 pm for a miniprep the next day and added to 5mL of LB + 25 mg/mL KAN + 1 mM IPTG and grown out in the 30C shaker

Another culture was started of GFP from the -80C freezer stock in 5 mL of LB + 100 mg/mL AMP for a miniprep. Both the ligations with GFP did not work, so as long as we are miniprepping and digesting we want to re-verify this part

8/1/2010

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

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 dense and when IPTG is added the copy number is over 100 so 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

• 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 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

flu primers

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

• General primer design
• Detailed primer design

8/3/2010

Minimal Media Recipes

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 (NA's)

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)
  • E. coli K12 in Bushnell-Haas minimal media supplemented with glucose
  • P. putida oil sands in Bushnell-Haas minimal media supplemented with glucose
  • P. flourescens oil sands 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 oil sands 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)
• 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 oil sands 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

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

8/4/2010

Culturing Bacteria in Naphthenic Acids (NA's)

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

• 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 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

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.

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

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).

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.

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

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

• dNTP's
  • 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
    • 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 85V according to the protocol on the wiki

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

8/5/2010

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:

• 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 started 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 digest it and run a gel to check anyways

Digest of pBAD take 2

the 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 because this showed to have less unspecific product (shown by the gel from yesterday)according to this modified colony PCR protocol. The volume of the reactions were increased to 50 uL to do 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 tried again tomorrow

Biofilm assay in minimal media

Cultures were started according to the following biofilm assay protocol to inoculate the microplate for 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

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:

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

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

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

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.

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.

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

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 of flu. The higher annealing temperature reduces the 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 disolve 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)

8/10/2010

Ann and Bryce

Transformation of Constituative 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 efficency of the procedure).

The biobricks we are transforming today are:

• J23100 (constituative promoter)
• I14018 (PCR purify to remove protomoter 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

• negitive control (comp cells only) on LB+100 mg/mL AMP
• constituative promoter on LB+100 mg/mL AMP
• negitive 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

and placed in the 37C 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.

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 and 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 37C incubator to grow up

at 9:00pm the cultures were minipreped 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 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 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

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 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

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. 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 (???).

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 DH5alpha 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

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 because 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 in 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 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 degrees 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, so a couple samples were a bit short (22L and another).

8/31/2010

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).

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)

In the Lab