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 labwork 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 spectrophotometrized.

(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 labwork 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.

(10:00 am) Na na na na na na na na BATMAN!

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

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

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

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

5. 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.

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

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

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

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

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

6. 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.

7. 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 realised what went wrong so it would work tomorrow. Just before finishing, 3ml of luria broth was innoculated 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.

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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 adapter and apply the pressure). As we didn't have an adapter, 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 coloumn 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 seperated 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, 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 transfering 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 resuspended 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 resuspend 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.

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 priscribed 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 recomended, we are spinning for longer to maximise 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 (*******) 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 centrefuge. 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 seperated it into epindorphs 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 tubesare then allowed to air dry for 20 minutes or until there is no liquid. before the addition of the TE buffer.

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