Team:Leicester/August2012

(9:30 am) Several members of the team are given wrong start times, so only 1 student arrived in the lab at 9:30, however with the work needed to be done all the rest of the team are quickly on their way.

(10:30 am) With everyone now in the lab it turns out a lot of the work planned for today has to wait until tomorrow. This is due to the ''Pseudomonas'' strains needed to be grown in a rich luria broth before we can spin it into a pellet and then run the DNA extraction.

(11:40 am) With all the bacteria placed in the 15ml corning tubes, and placed in the orbital shaker all of today's lab work is complete. Now the team is going to finish the recording for the rockethub video as some scenes need to be retaken, then do some individual research/work.

(13:30 pm) After consulting with a supervisor and having a long meeting over lunch the team has decided several directions that is needed to go in. One member is testing the cooling times of a water bath that will be used in the hybridizing process of making our DNA libraries, and to start selecting out of the DNA genes that are in both our Pseudomonas and the NB26 strain that definitely degrades polystyrene. A couple of members are looking through protocol and methods to use with the DNA extraction.

(9:30 am) An early start finishing all the protocol and work needed to start making the DNA libraries.

(10:00 am) ''E. coli'' is placed into 7ml of LB media and put into the warm room to grow overnight and be used as the test tomorrow to check the DNA extraction process before using the ''Pseudomonas'' species.

(11:30 am) The protocol for tomorrow's practicals is determined, for the test of the DNA extraction, as well as dilutions to check the growth so there is a measurable amount that the ''Pseudomonas'' can be compared to, so in future to measure the amount of ''Pseudomonas'' the team has it can simply be spectroscopically analyzed.

(4:00 pm) Made the ''E. coli'' into a 50ml broth to grow overnight for the dilutions and spectrophotometry.

(9:00 am) Action stations. All lab work is go. Today there are 2 experiments going: the DNA extraction test and the dilutions to test the method works. Once these are done, the team knows what works and doesn't work for the real experiments on ''Pseudomonas'' early next week.

(10:30 am) The team splits into 2 groups and goes to do the tasks.

The half doing the dilutions to make the growth curve left our dedicated bit of lab space and headed into one of the supervisor's labs with a good spectrophotometer and space to work with the bacteria.

Protocol:

1. Take a reading of the overnight broth.

2. Innoculate a new conical flask of 49.9ml luria broth, with 0.1ml of overnight culture.

3. Put 1ml of culture into a spectrophotometer and take a reading at 600nm. Record this as time = 0. Start the timer.

4. At same time, take an aliquot of 100ul and put in eppendorf marked as time 0, 10^-1 dilution. Add 900ul of buffer and mix, then take 100ul from this solution and add to next eppendorf, marked time 0, 10^-2 dilution and so on to 10^-9.

5. Pipette 100ul of dilutions 10^-4 to 10^-9 onto similarly labelled pre-prepared agar plates and spread them.

6. Repeat step 2 every 40 minutes and step 4 every 2 hours (3 divisions of E. coli) until the end of time.

7. Leave plates to grow, then count the number of colonies growing on the most concentrated plate where colonies are distinguishable. Multiply up to the true concentration, then multiply by 10. This is colonies/ml.

8. Draw graph to see correlation and check against known correlation to see if it has worked.

(16:30 pm) The dilutions and CFU count part of the experiment is finished, there is 6 hours of readings for the CFU count as well as plates for 0, 2 and 4 hours.

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The only thing needed to be done today is help the technician gave the team. This involved producing ''E. coli'' overnight cultures for the DNA extraction and digest.

(9:30 am) Today the team is going to extract the genome and start the DNA digest of the ''E. coli'' to test the DNA extraction kit and procedure that the team has identified to use.

This is a big step that if works, can then be applied to the ''Pseudomonas'' strains that are growing. So that once the NB26 strain arrives it can be compared straight away to give an idea which genes could be involved in the degradation of polystyrene and to help start narrowing our search.

The overnight cultures of E. coli were then analysed using the spectrophotometer to give an initial bacterial cell count before the digest.

(12:30pm) The results for Friday's experiment is back. Unfortunately the data that was collected is not enough to create a graph to simply show us what the bacteria cell count is from reading an absorbance.

The new protocol for today's experiment that will tell the team this data is:

1. Take a reading of the overnight broth.

2. Inoculate 49.9ml of broth with 0.1ml of the overnight bacteria colony. Mix, start timer, and immediately do 3 and 4.

3. Put 1ml of culture into a spectrophotometer and take a reading at 600nm. Record this as time = 0. Start a timer.

4. At same time, take an aliquot of 100ul and put in eppendorf marked as time 0, 10^-1 dilution. Add 900ul of buffer and mix, then take 100ul from this solution and add to next eppendorf, marked time 0, 10^-2 dilution and so on to 10^-7.

5. Pipette 100ul of dilutions 10^-5 to 10^-7 onto similarly labelled pre-prepared agar plates and spread them.

6. Repeat steps 3-5 every 40min (1 doubling of E. coli) (labelling time appropriately) until the end of time.

7. Leave plates to grow, then count the number of colonies growing on the most concentrated plate where colonies are distinguishable. Multiply up to the true concentration, then multiply by 10. This is colonies/ml.

8. Draw graph to see correlation and check against known correlation to see if it has worked.

(13:30pm) After finally finishing all the reading up and gathering of ingredients, the DNA extraction process is finally started. Protocol is as mentioned in the QIAGEN Genomic DNA Handbook.

Almost as soon as it was started the first few sections were complete and the cells needed to be left in a 37oC water bath for a while so the team members had to wait again. And then had to wait again after adding different buffer while the cell extract was left at 50oC.

(15:00) The current step is using the genomic tip protocol to extract DNA. Unfortunately, the flow rate is very slow so this may take a while until the DNA can be washed and eluted.

(17:30) Today's refinement of the growth curve experiment worked. Well at least the protocol did, the actual experiment failed but after talking to the supervisor in a group meeting, the team realized what went wrong so it would work tomorrow. Just before finishing, 3ml of luria broth was inoculated with a single colony of ''E. coli'' as well as the plates being made up.

(8:00 am) A very early start for part of the group working on fixing the dilutions experiment. In for 8am, and started at 8:30 for a long day's practical.

(14:30 pm) The final set of plates and spectrophotometry readings have been taken. Now they can grow overnight to see if it works.

(9:30 am) The plates had grown, but unfortunately they had all grown a ridiculous amount. It turns out that the luria broth had been contaminated with some bacteria. The group didn't realize this as it was thought some of the nutrients had suspended like what had happened to our minimal media beforehand. This means another experiment must be done to see if it can finally be done right.

(11:30 am) The final dilutions experiment was started, but it needed time to grow out of lag phase. Just before lunch it still hadn't started to grow so the group was starting to despair, but after a long lunch break it had finally started to grow.

(14:30 pm) The final series of plating and spectrophotometry has started, only time will tell if it is finally right.

(11:00 am) It worked! The final dilutions experiment came back with close to expected results so the plates could be counted and a graph made to show the cell count against absorbance.

While the lab leader was away the iGEM team carried on with business as usual. However it was a different kind of business as we decided to work on the Rockethub video, as it was getting late on in the time we had left to complete the video with spare time to edit and so forth.

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Chris was working from home on the Rocket hub site, then had a prior engagement.

(11:00 am) Continuation of the DNA extraction. Finishing plating up all the CSE kits onto minimal media and polystyrene mix.

(12:00 pm) All the CSE kits are plated and incubating in the warm room ready to be analysed and hopefully come up with a positive result.

(16:00 am) iGEM Weekly meeting, after this Chris prepared the overnights ready for starting the next Genomic tip , while Dr badge and Will prepared the lab ready for tomorrow after the leak. result.

Will and Chris, Setting up to run the gel tomorrow, as well as finishing off the genomic tip number 2 prep. This included Chris and Dr Badge trying to improvise a method to generate +ve pressure on the tip to a rate of 20-40 drips per min, as it was taking in excess of 24 hours for 10ml to flow through the column we thought this was necessary (protocol says for slow rates to use a adaptor and apply the pressure). As we didn't have an adaptor, or syringe plunger that was the correct size, we improvised, testing out with the used column 1 different ways to apply the pressure, be that by adding a cut down syringe to the top of the column which then could accommodate a plunger, and the other method of using a oversized plunger rubber, using a thumb to press it into the top of the column to create the pressure needed. The second method was used this time to finish off the column as there was only about 2ml to run through. After this we prepared the samples to load onto the gel tomorrow. Once the column was finished, we separated the eluted DNA equally into 24 eppendorf tubes and span for 11 minutes at 13,000G. This was an alteration to the protocol by QIAGEN as it stated at least 5000G in the falcon tubes, however as our centrifuge can't do this with the 50ml falcon tubes we separated it into eppendorf tubes which could then be span faster. 5000G was the minimum so we span them at the max for the centrifuge, making sure the hinge was pointing outwards so to know where to look for the pellet. After this there was no large visible pellet, and so we proceeded as if there was one on the orientated side of the eppendorf away from the hinge, making sure to not disturb the tiny amount of DNA that could have been there. After a second spin to remove more liquid, we then added 500 micro litre of the 70% ethanol to wash the DNA of any salts using the same 500 micro litre transferring it between 5 of the tubes at a time as the total volume of ethanol to be used was 4ml. The supernatant is then removed and the eppendorf tubes were then allowed to air dry for 20 minutes. The DNA was then re-suspended in 50 micro litre per eppendorf in TE buffer ( PH 8.0 ), placing all of the eppendorf tubes in the 55 degrees hot block for the hour dissolving stage to re-suspend the DNA. Once this was finished we recombined the DNA into a falcon tube and prepared the aliquots for the Gel electrophoresis For the samples, we did a 4x dilution of each aliquot, as well as the pure DNA, just in case there is a large amount of genomic DNA in the samples. These are stored in the 5 degrees room ready for the morning. Phil, Nathan and Tony The experiment for the rate of doubling within ''P.aeruginosa'' was started, the experimental details were as follows. 1) Inoculate 49.5ml broth with 0.5ml of concentrated strain to give a 1% dilution (stock 1). Leave in warm room for 135min. 2) Take 5ml of stock 1 and inoculate into 35ml of broth. Start timer, take spectrophotometer reading at 600nm (zeroed with broth) of 1ml of both stock 1 and the further 8 fold dilution (stock 2). 3) After 5min, take a further reading of stock 2. Repeat for 135min, at which point the spec reading should equal the stock 1 reading at time zero (three doublings at 45minutes each).

Will and Chris,

(9:00 am) Chris loaded the dye upon the gel electrophoresis and it is currently running the aliquots taken along the genome extraction of E.coli to make sure we have been doing it correctly. There seems to be very low yield from the maxi prep so are trouble shooting the problems with the tip e.t.c and altering some of the steps from the prescribed QIAGEN genome extraction kit.

(11:15 am) Boiling the RNase for 10 minutes to make sure that it re-conforms to the RNase rather than DNase which is possible after a long period of time. This may have been the case with the previous 2 genomic tips, so trying this to see if we can gain a higher yield this time. Gel is still running and is now about half way. Will is now using the nanodrop spectrophotometer to see if there is DNA in the aliquots and the pure DNA elution.

(12:00 am) Genomic tip 2 gel results were blank, (image to follow) so we're thinking it was the RNase as there was no DNA in even the aliquot 1 from the gel. Markers and our mini prep showed the DNA ran as such. Now we are preparing the cultures Chris made two nights ago for another Maxi prep with the E.coli, however with the boiled RNase and other alterations to the method. to start we are pelleting the bacteria, OD600 3.25 scaled from a 10x dilution, so we have 2.6x10^9 cells per ML, the max for the prep is 1x10^11 cells so we are using 28ML of our culture, working out at 7.28x10^10 cells which is in the limit. the cultures have been balanced equally and are now spinning in the centrifuge at 2600G which is as high as the centrifuge will go with this size falcon tubes, as this is slightly under the 3000G recommended, we are spinning for longer to maximize the size of the pellet. Once the supernatant has been removed, we will re-spin to make sure any remaining liquid has drained to the bottom to then pipette this off to make sure there is no supernatant left.

(14:30 am) Lysate is now in the 50 degree incubation stage to digest the proteins from the solution so that they can flow through the column. This has now been going for 45 minutes and has yet to become as clear as before, due to this we are going to incubate for another 30 min then spin at 4 degrees for 20 minutes which maybe due to having cells that are intact. We then want the clear supernatant to go into the tip. While Will is doing this, Nathan has made up more of the 0.5% TBE buffer solution for the gel tank while Chris is making the agarose and preparing the gel tray, This was done by following the GENIE video which can be seen on youtube at ([http://www.youtube.com/watch?v=wXiiTW3pflM]) the agarose is currently in the Hybridiser cooling to 60 degrees so it can then have the Ethidium Bromide added and be poured into the gel tray. Results from the Nano Drop Spectrophotometer were 2.5ng per micro litre ( from read out), A260 = 0.051 A280 = 0.021 Ratio = 2.43 which is quite high as it should be between 1.7 and 1.9 which means that we may have had DNA with a mixture of RNA but running the gel shows that there was no bands of DNA so it was most likely digested... Hopefully the boiling of the RNase to get it to refold should fix this problem as well as doing the other additional steps Dr Dalgleish suggested.

(16:15 am) The cell lysate had still yet to become clear after the further incubation, so we span for what ended up being 35 minutes at max speed for the 50ml falcon tube centrifuge. There then became a large fluffy mass of protein and cells that was at the bottom ( however not forming a pellet) which is making it hard to remove the supernatant that contains the DNA. We are now trying to remove this by passing the lysate through a 4 micron syringe filter

(17:15 am) Protein mass was removed and the lysate was passed through the genomic tip. The flow rate was much faster than the other two preps, being within the 20-40 drops per min as stated by the kit, with a slight positive pressure from time to time. We ran through the kit, and have ended up getting to the precipitation stage

(11:15 am) Eluted DNA from yesterday is now spinning down the centrifuge, we separated the eluted DNA equally into 24 eppendorf tubes and span for 11 minutes at 13,000G . This was an alteration to the protocol by QIAGEN as it stated at least 5000G in the falcon tubes, however as our centrifuge can't do this with the 50ml falcon tubes we separated it into eppendorf tubes which could then be span faster. 5000G was the minimum so we span them at the max for the centrifuge, making sure the hindge was pointing outwards so to know where to look for the pellet. After this there was no large visible pellet however there was at least something this time that looked like a pellet orientated on side of the eppendorf away from the hinge. After a second spin to remove more liquid, we then added 500 micro litre of the 70% ethanol to wash the DNA of any salts using the same 500 micro litre transferring it between 5 of the tubes at a time as the total volume of ethanol to be used was 4ml. The supernatant is then removed and the eppendorf tubes are then allowed to air dry for 20 minutes or until there is no liquid (Before the addition of the TE buffer).

Did the 1st pseudomonas dilution experiment.

(11:30 am) Not much to do today with most of the group were at Google in London, giving a presentation on the iGEM project.

However that being said, the remainder of the group was just left to count colonies of the previous days' work. There was an explosion of colonies, so the experiment would have to be repeated on Tuesday, allowing the overnight broth to be made on Monday.

As well as running the DNA prepared yesterday. This caused a bit of the problem as the gel was such a low percentage it broke very easily, but eventually it was set and it could be run.

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will and Chris: yesterdays preparations for our mini prep allowed us to get into full swing with lab work. As working to extract the genome from aueroginosa we had to work in a different lab that was certified to handle pathogenic organisms. (9:30) moved all equipment necessary into other lab, set up water baths for the incubation’s at 37 and 65 degrees that will be needed later in the day. spinning down our aueroginosa to get a pellet of cells we re suspended them in p1 buffer before adding the NaCl and lysozyme to lyse the cells. incubated at 37 degrees for 30 minutes the solution should go clear and viscous. solution has gone clear and viscous... success. Addition of 10% SDS and proteinase K to the solution, this will strip the DNA of bound proteins and remove secondary and tertiary structures with in the proteins. incubation for another 30 minutes to allow the reagents to act on the DNA in solution.The addition of preheated CTAB, as a cationic surfactant, acts as a buffer for our DNA extraction and later when running our gel prevents the generation of fuzzy bands. Another incubation period is required for the reagents to react. After this the cholorophorm:isoamylalcohol isolates nucleic acids (RNA and DNA) in a separate layer after they have been centrifuged at 13000 rpm for 10 minutes. The visible barrier between the two layers or interface, separates the nucleic acids in the top layer dissolved in aqueous solution and the lipids and proteins dissolved in phenol and chloroform in the bottom layer. The top layer is the area we want to extract and transfer to another tube. This process is tricky as it is hard but also important to not disturb the inter phase layer and thus we must prevent the top and bottom layers remixing. We removed as much as possible by hand, but being unable to remove all of the top layer it was decided to repeat the chloroform extraction step/remix, re add chloroform:isoamylalcohol and re-centrifuge. After this we removed all the remaining top layer supernatant and proceeded with the next step. The addition of the isopropanol acts as as a way to precipitate the DNA out of solution into a pellet by centrifugation as DNA is insoluble in the isopropanol. The isoproponal can then be removed and air drying the pellet will ensure complete removal of the isopropanol. The re suspension in TE buffer will stabilize the DNA in solution, ready to run our gel. but before our gel is run a nano drop will be performed to ascertain the nucleic acid concentration and absorbance values at 260 and 280 NM plus an absorbance ratio calculated from these values.

Reran the pseudomonas with a slightly altered protocol. -8 rather than -7.

(9:00) will: went to enquire about using the nano drop, asked DR Dalgleish for assistance and instead showed me how to use it, resulting in me now being proficient in use of the nano drop, although a supervisor is required to watch over me using this delicate piece of equipment. Having run the nano drop our results look very promising, our A260/A280 ratios are a little high averaging 2.10, perhaps due to RNA contamination. But the RNA can be removed later by an Rnase treatment if it is very contaminating. A gel will be run to ascertain the amount of RNA contamination. Our absorbency curves were text book examples of what you expect when looking for DNA which was very pleasing.

(10:30) will: the samples were prepared. Each sample was 15 micro litres in size with concentration of 200 ng of DNA in total. With the varying DNA concentrations in each of our 5 preps, ascertained form the nano drop, the amount of sample loaded into each well varied and as a result the distilled water added also varied, this was to ensure that equal volumes over all were added into each well to be run. The samples were loaded by Luke. The size marker we are using is lambda Hind 111 (23kb). Due to the high molecular weight of DNA we aren’t expecting our sample to run down the gel very far. The presence of RNA should appear as a blob at the bottom of the gel, if there is a lot then we now there is very heavy contamination and the RNA can be removed and the gel re run. Alternatively if there is contamination but it is very slight then we can remove the RNA when we perform our DNA digest. If there is no contamination from RNA then our high readings on A260/A280 ratio will have some other explanation.

(13:46) gel finished running, the bands are visible but faint on the photo. There seems to be no contamination from RNA, therefore the next plan of action is to get a higher concentration genome extraction yield, using the following protocol:

Morning - checked for colonies and it was clear there was error. Most -7 had more cells than -6. Spreading problems continued as uneven spread formed massive groups of colonies, unable to be counted easily.

Afternoon - reran experiment with plate spinner, finishing just at 6. Slightly altered protocol again - spreading 300ul, spinner.

CTAB

need to take the DNA which is ~500 micro litres in TE, add equal volume of isopropanol mix thoroughly but gently. spin for 10 min at 13,000 RMP / as hard as possible, hopefully there will be a pellet of something. pipette of supernatant not disturbing the pellet. Re-spin to get as much of the isopropanol off, then air dry. then resuspend in 100TE to the first tube to dissolve the pellet. when this has dissolved, take this and add it to the next tube, each time making sure the pellet has dissolved, mix, tap, but don't pipette, need to get the pellet into solution before you go into the next tube. carry on through the 5 tubes, until you have all of the pellets resuspended in the same volume of liquid.

spec on the nano drop to check the concentration. blank = TE then run on gel .

(15:20) Scraped a colony from C.S.E 01#502 onto a slide and examined it under a microscope and as it surrounded polystyrene beads it was intriguing. Strategised plan is too plate out onto 2x luria agar and 2xminimal media plates 2x 5% sprinkled poly plates. The plates will be inoculated and one 5% sprinkled poly, one minimal media and one luria agar placed in the 37 degree room. And the other three inoculated plates left at room temperature. We will see in the morning which has grown better.

(16:40) Chris had made a new 1% agarose gel, using the award winning GENIE vid available at [http://www.youtube.com/watch?v=wXiiTW3pflM] . Once this had set, it was then loaded into the gel tank and filled with 0.5X TBE buffer solution. We were getting low on this solution so diluted some 10X TBE into 2litres of 0.5X TBE and one litre of 1X TBE. The samples were then prepared with 3 micro litres of 5% loading die, and pipetted by Chris into the gel which is currently running. the lane organization was

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15 micro litre marker

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5 micro litre 1kb DNA ladder from ThermoFisher Scientific

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15 micro litre Sample DNA

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5 micro litre 1kb DNA ladder from ThermoFisher Scientific

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15 micro litre marker

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(17:23) Starting to plate out the 01#502 onto the three different types of media to be placed in the two different conditions. Fingers crossed tomorrow we have our 'positive' growing well to then use this. As well as that, we are going to photograph the 01#502 plate to see if we can get a detailed photo from the microscope. Chris has taken one with his phone at the moment which is ok, however i think we can get much better resolution with a actual microscope camera.

Need to do a CTAB for ''p.Aeruginosa''

(9:07) Chris has put the gel is back in the tank after being in the cold room wrapped all night and is running it at 100 volts. Will is currently preparing for the next CTAB extraction by getting the reagents ready. Phil is currently looking over the CFU counts from yesterday which there seems to be a problem with... ( very few colonies), wondering if this is a problem with the technique, or in the calculation of the doubling time, either way, it isn' working correctly so need to try and trouble shoot. Will is now melting agar for making new plates for another CFU of ''p.Aeruginosa'' and is going to do a sectro reading for the overnight culture of ''p. aeruginosa''making sure we get a significant reading ( between 0.05 and 0.3 ) and then scaling up to the OD600 for 1ml of culture.

(9.40) spectro reading came in after scaling up at 5.7 which is quite high, however we won't know how many cells we have until we can get the CFU to work however it is many more cells than before, so fingers crossed we can get some chromosomal DNA out this time. Luke is currently looking at the enzyme modification ***** INSERT TEXT HERE LUKE****

Yesterday's experiment failed, the Pseudomonas decided not to grow at all this time apart from the 240 mins -6, and -7 plates. Now running a plate test to see if the failed yesterday experiment was down to faulty agar or just the cells where diluted out as the optical density wasn't that high.

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