Team:Leicester/September2012

(11:00) Nathan and Chris went to the lab and started to work out the concentrations of the 16s extracted DNA to run on the gel, ready for sequencing. We don't want to use up to much of our DNA so needed to work out the optimum amount to run on the gel.

(14:00) Calculations done, took some time as Chris had a meeting at 12 for a hour and a half. Had it checked and now are going to get ready to run 2μl of each of our sample, and 4μl of sample number 2 which has a lower ng/μl reading. As we have only a small amount of DNA we are to vary the amount of the markers for different concentrations of DNA to work out the concentration of our samples to make sure the nano-drop was accurate. After this we can get ready to sequence the DNA to find out what the bacteria is.

(15:45) Nathan loaded the gel and then put the gel lane organisation
x
x
marker 4μl
2 (4μl)
3 (2μl)
4 (2μl)
marker 2μl
5 (2μl)
6 (2μl)
marker 1μl
x
x
x
x
Gel is now running, and will be complete in 1 & 1/2 hours time.

(16:30) Group meeting to bring our supervisors up to speed with the project progress, and discuss terms and action plan for the following day, as well as a look into future plans.

(17:30) Meeting finished early so that Chris could stop the gel and then transilluminate to get a gel photo, allowing us to work out the concentration of the DNA more accurately and thus allowing us to work out how much to use for the sequencing to be done tomorrow.

(09:00 am) Some colonies from the growth curve experiment could be counted, but in general there was too much growth.

(09:15 am) The group worked on the wiki for the rest of the day writing the past weeks work in, making sure all the details were correct and in the right order, and then attributing the members as required.

(9:00) Chris and Luke discussed the pathways for the project, before a meeting at 11 with Dr Dalgelish and Dr Badge to discuss what to do next. We are hoping at this stage, although late in the competition, to try and engineer a BioBrick. We are looking at the TodX, TodC1&2, TobA&B and other genes in the operon for toluene degredation which we think is also the pathway for the polystyrene degradation.

(12:00) After the meeting Chris and Luke started looking though the genomic sequences and BLAST searching for bacteria that had proteins similar to the ones above to PCR out; though Pseudomonas aeruginosa seems to have nothing, Pseudomonas putida F1 has all of the genes.

(15:00)Dr Badge and Chris set up the PCR reaction of the 16S to increase the amount of DNA in the forward and reverse directions (in separate tubes) ready for sequencing tomorrow. Chris then plated out the Yellow colonies, Orange colonies, and re streaked the 01#502 so we have fresh colonies ready to prepare them for storage as we are coming close to the end of the project.

(16:00) Chris is now writing up the wiki while luke is looking through BLAST searches again to find proteins. Phil is currently getting spec readings for the mmp broth with the 01#502 which came out at mixed culture 0.334, orange culture 0.6 at OD600 blank being the mm, which looks good as there is no other carbon other than the polystyrene.

(09:00 am) An early start with no set experiment today. Two of the colonies were plated out to see if they are two forms of the same bacteria or simply two separate species. This required plates being made, then individual colonies picked out to streak them. Once finished it is a day working on modelling, writing protocol and other computer work before checking the results of the plates in the late afternoon.

(16:00 pm) Some spec readings were taken of the MMP broth, with the blank just being MM broth. At 600nm wavelength the results came back for the mixed culture as 0.334, and the orange culture as 0.6.

Nathan editing the wiki once more.

(14:00) Meeting to discuss important matters before going to Amsterdam. Everyone was brought up to speed with what each individual section of our project (modelling, lab work and chemistry) has done, found out and achieved. Job roles were assigned to each person to complete in labs and on the computer modelling.

(16:30) Luke has prepared more polystyrene minimal media plates ready for plating out some colonies from CSE kits, to see whether more positive results can be found from other samples.

(17:00) Will is making more minimal media from minimal broth, to make more polystyrene minimal media plates ready for the CSE kits and potentially the Pseudomonas putida strains we may be getting shortly.

(17:05) Chris setting up ready for the boilate with a sample of our unknown bacteria to extract the DNA, ready to set an overnight PCR reaction so that we can sequence the 16S ribosome later on this week.

(17:20) Will has now taken over doing the boilate so that Chris can set up the master mix for the PCR reactions. As we are doing 14 PCR reactions we need to make up 270μl of the master mix which is enough for aliquoting 18μl into 15 reactions. reaction mixture list:
171μl PCR H20
60μl HF buffer
6μl dNTP's
15μl Primer A 28f AAGAGTTTGATCCTGGCTCAGA
15μl Primer B 519R GWATTACCGCGGCKGCTG
Template DNA to be added later as it is not PCR clean
3μl DNA Pol
These reagents were added then as the DNA Pol was in glycerol the tube was mixed and spun for a second to remove bubbles. dNTPs cannot be freeze thawed so whatever was left was thrown. After this, in the hood Chris took 18μl aliquots of the master mix into 9 separate PCR eppendorfs and prepared the -ve control while in the PCR hood with the PCR clean H2O. The tubes were then taken to the bench where the DNA and diluted bacteria were added, and the +ve and -ve controls (see gel lane organization) all samples were added at 2μl to make a final volume of 20μl.
PCR Cycle program used was iGEM16S which is as follows:
98 degrees C - 5 min
''98 degrees C - 30 seconds''
''50 degrees C - 30 seconds''
''72 degrees C - 2 minutes''
72 degrees C - 5 minutes
15 degrees C - Forever ( this is for the end of the reaction)

(9:00) Chris, Will, Emily and Luke in lab, Anthony and Phil in computer lab. Luke is writing the protocol page on the wiki, while Chris is making a 2% gel ready to run the PCR reaction mixture. Will is currently trying to track down some of the P. putida F1 strain which we may have located. Luke is writing up some of the protocol in the project section of the wiki.

(9:40) Chris has poured the gel which is setting and is now going to prep the PCR reaction mixtures adding 5μl of loading dye to each of the mixtures, ready for them to be run once the gel has set.

( 11:16) Chris has now loaded the gel and is running it at 120 volts. Lane organisation:
1) +ve control 10ng P. aeruginosa DNA from the Maxwell prep
2) +ve control 10ng P. aeruginosa DNA from the Maxwell prep
3) Neat orange culture DNA
4) x10^-1 dilution Orange culture DNA
5) x10^-2 dilution Orange culture DNA
6) x10^-3 dilution Orange culture DNA
7) x10^-4 dilution Orange culture DNA
8) Neat Yellow culture DNA
9) x10^-1 dilution Yellow culture DNA
10) x10^-2 dilution Yellow culture DNA
11) x10^-3 dilution Yellow culture DNA
12) 5μl Marker 100bp thermo scientific
13) -ve Bench H20
14) -ve Hood PCR H20
Luke has prepared the Sau3AI partial digest. This time the experiment will only run for 30 minutes as from the last test the only lanes which were digested enough were the times 5-15 minutes. A "no enzyme" control will also be run.

(12:30) The sequencing results of the 16S ribosomal DNA came back, so Chris and Luke did a BLAST Search to find the genus of the bacteria cultured from the 01#502 CSE kit, which turns out to be a Pseudomonas of unknown species.

(13:20) Chris stopped the gel and went down to the transilluminator with Emily to get a gel photo (image to follow). The x10^-2 and lower dilutions showed no bands, but the 0 dilution and x10^-1 dilutions worked well. The PCR was re-run over night with the x10^-1 orange dilution and the 0 yellow dilution with 5 of each to make sure that we have enough DNA to Sequence, as the amount recovered from this gel may not be enough for the sequencing. This is being done as there are two different colour bacteria growing, to see if these are two different species of bacteria, or the same one with a different morphology of colony. Chris and Emily are now going to do the QIAGEN gel extraction of yesterday's PCR to see how much can be recovered; more will be amplified over night. Will is making a gel for Luke's Sau3AI digest, which has now finished and is ready to be run.

(15:10) Chris and Emily have now cut the gel and have removed and weighed the samples of DNA, Chris is now adding the QG buffer at x6 the amount in μl of the amount of agarose there is. Emily is now going to put the samples in the 50 degree incubator and vortex every 2 minutes to dissolve the agarose.

(15:30) Chris and Luke just realised that the Sau3AI digest was carried out at 37 degrees again rather than at the room temp we were going to try to see if this produced better bands. So we will have to re run this experiment, however we are still going to run the gel to see how it went and confirm the need to reduce the activity of the enzyme. Will is speccing the experiment that nathan set up on the 31st, after 2 day's extra growth; mmb with 5% poly, OD600 mixed = 0.053 at a 10x dilution so 0.53 Orange is 0.072 at a x10 dilution so 0.72 which is higher than before, so it looks to be that the bacteria is growing in the broth with no other carbon other than the sugar beads at 5% concentration. To confirm this will is re doing the experiment with more controls totaling at 8 tubes (Will to put protocol in here).

(16:00) Will is taping the shaker to the bench to prevent it from moving. Tony is loading up a gel using Luke's Sau3AI digest from earlier. Chris and Emily are extracting DNA from the earlier electrophoresis using a QIAGEN QIAquick Gel Extraction Kit. Luke is writing up protocol for some of the experiments we've done so far in the projects.

(16:30) Chris is now doing the boilate for tonight's PCR of the 16S yellow and orange colonies; only the 5 neat yellow cultures and 5 x10^-1 orange cultures will be PCR'd as these seem to be the best, though it looks like we don't have enough DNA to sequence.

(17:15) Emily is finishing off the boilate, doing the centrifuge step, removal of the supernatant and doing the dilutions while Chris is in the PCR hood setting up the master mix.

(17:45) Chris is just adding the DNA to the samples ready to put them in the PCR machine, Program to be used is the iGEM16S which is as follows:
98 degrees C - 5 min
''98 degrees C - 30 seconds''
''50 degrees C - 30 seconds''
''72 degrees C - 2 minutes''
72 degrees C - 5 minutes
15 degrees C - Forever ( this is for the end of the reaction)

(9:00) Chris, Will and Luke in lab, Phil in computer lab. Will started to make a new 2% gel ready for the PCR samples which Chris is preparing, Luke is getting ready for the Sau3A1 digest at room temp

(10:30) Luke has almost finished the Sau3A1 prep and Chris is re-making one of the gels. Will is making another 2% gel ready for the "Gelception" gel and is waiting on someone to help with the Nanodrop of the gel extraction from yesterday.

(12:10) After having a lot of problems with gel making today, Chris is finally loading the PCR gel from last nights PCR reaction this time loading as much of the sample as possible being 22ul. This will be ran for 2.5 hours at 120volts to make sure the bands are well separated for the gel extraction. Sau3A1 prep is finished and the samples have been put into the freezer ready to be run on Monday.

(13:00) Working lunch break for Chris and Luke, looking at the primer designs for BioBrick.

(15:36) Luke is now designing the primers ready to order for Monday morning. The gel is now finished so Chris, Luke and Emily take that down to be transilluminated, as ''Pseudomonas'' species' fluoresce under UV.

(16:00) None of the plates fluoresced under UV, though this may be due to the wavelength different wavelengths will be tried. Will is now wrapping the PCR gel ready for gel extraction on Monday. As the Nanodrop results were low for yesterday's gel extraction from the PCR at 4.8 for 0range 5.1 for mixed and 3.2 for yellow when the two extracts were combined, Chris is going to do a PCR of these samples to increase the amount of DNA. The "Gelception" gel is to be run on Monday as there isn't enough time to run it today.

(16:30) Luke has finished the primer sequences and has sent them to Dr Badge to be checked and ordered. Chris is now setting up the master mix for the PCR in the PCR hood as we are doing 13 PCR reactions, we need to make up 270ul of the master mix which is enough for aliquots of 18ul in 15 reactions. Reaction mixture list:
171ul PCR H20
60ul HF buffer
6ul dNTPs
15ul Primer A 28f AAGAGTTTGATCCTGGCTCAGA
15ul Primer B 519R GWATTACCGCGGCKGCTG
Template DNA to be added later as it is not PCR clean
3ul DNA Polymerase
These reagents were added then as the DNA Pol was in glycerol the tube was mixed and spun for a second to remove bubbles. dNTPs cannot be freeze thawed so whatever was left was thrown. After this, Chris put 18ul aliquots of the master mix into 9 separate PCR eppendorfs and prepared the negative control while in the PCR hood with the PCR clean H2O. The tubes were then taken to the bench where the DNA was added, and the positive ''aeruginosa'' DNA and negative bench H2O controls all samples were added at 2ul to make a final volume of 20ul.
PCR cycle program used was iGEM16S which is as follows:
98 degrees C - 5 min
''98 degrees C - 30 seconds''
''50 degrees C - 30 seconds''
''72 degrees C - 2 minutes''
72 degrees C - 5 minutes
15 degrees C - Forever ( this is for the end of the reaction) This time instead of loading bacteria from a boilate reaction Chris added in a 1/100 dilution of the three different gel extractions at 2ul to each tube to amplify the amount of DNA in these samples. Luke prepared the 1/100 samples by taking 1ul of the DNA and adding in 99ul of TE buffer. Reaction organisation ( CHECK ON CHRIS' SHEET TO SEE IF THIS IS CORRECT)
1 = positive ''aeruginosa'' control
2 = orange 100x dilution
3 = orange 100x dilution
4 = orange 100x dilution
5 = yellow 100x dilution
6 = yellow 100x dilution
7 = yellow 100x dilution
8 = yellow 100x dilution
9 = mixed 100x dilution
10 = mixed 100x dilution
11 = mixed 100x dilution
12 = negative control bench
13 = negative control hood The reason why there are four reactions for the yellow is that it had the lowest ng/ul reading so thought this would be useful. As there was a problem with the dilutions in the boilate from Wednesday, we are running multiple reactions for each of the extractions in case one doesn't work.

Chris is meant to be having the week off, however is working from home today on the primers using in silico PCR and BLAST searching

(9:00) Nathan, Will, Luke and Anthony in lab. Nathan started preparation of yet another 0.7% gel, Will and Luke found DNA markers - "HyperLadder 1"

(10:30) Luke started loading a 2% agarose gel with DNA recovered from a previous gel from last week. The lane order is:
5µl DNA HyperLadder 1
x
4µl 100bp DNA ladder
x
20µl of DNA recovered from PCRed mixed colonies
x
2µl DNA HyperLadder 1
x
20µl of DNA recovered from PCRed Orange colonies
x
20µl of DNA recovered from PCRed Yellow colonies
x
1µl DNA HyperLadder 1
x This gel will be run at 120 volts

(11:00) Luke pours the 0.7% gel ready for running the Sau3A1 digests.

(11:50) Luke, Nathan and Tony go to transilluminate the gel run with recovered DNA from a previous gel, however, the bands have moved little, so we decide to run it for another hour.

(12:15) Tony loads Sau3A1 digests into wells on the set 0.7% gel. The lane organisation is:
x
DNA HyperLadder 1
x
DNA digest without enzyme
DNA digest after 0 minutes
DNA digest after 5 minutes
DNA digest after 10 minutes
DNA digest after 15 minutes
DNA digest after 20 minutes
DNA digest after 25 minutes
DNA digest after 30 minutes
x
DNA HyperLadder 1
x This will be run at 100 volts

(13:00) Luke and [insert name here] transilluminate the recovered DNA gel again, where the bands are a lot more spread

(13:20) Luke and Will check Sau3A1 digest- we decide to leave it in for longer, though the loading dye appears to be running slightly quicker at one end of the gel

(13:30) Luke looks through the primers he helped to design on Friday, that Dr Badge has given a few tweeks, to add degeneracy to make them more likely to PCR out the genes we want to extract. Using BLAST searches of the FASTA data, it is apparent that the sequence in P. putida, regardless of strain, is remarkable conserved, with only one or two bases needing any degeneracy at primer binding sites. These modified primers are sent off to Dr Badge well before the deadline, so will hopefully be made up and sent to us before the end of the week, so Chris can start the PCRing out of genes we’ll hopefully be making into Biobricks.

(14:45) The Sau3A1 digest gel’s loading dye is a couple of inches off the end, so Luke and Will go to transilluminate it. We find that, despite part of the gel appearing to run more quickly, we have a decent digestion curve, showing a downward trend in fragment length as the time of the digest increases.

Luke and Tony attempt to write this notebook entry several times later that day, but each time, the website logs us out, and deletes the entry

(9:00) Chris, Nathan, Luke and Will came into labs, while Phil was editing the Wiki at home. Day started with Will getting the p.putida streaked out onto LA so we can get single colonies and also getting the p.putida strains into LB so we can do the Maxwell prep tomorrow to extract DNA from these strains. Chris then started to prepare to run the 1/100 dilution PCR on a gel which has been kept in the fridge. Luke is currently righting up the Wiki for yesterday and Nathan is making a 2% gel ready to run the PCR reaction

(11:00) Luke is preparing polystyrene 'sugar' for a polystyrene-propane experiment, to see whether propane, which is an expanding agent in the expansion of polystyrene could have caused the mystery Pseudomonas species to grow, rather than the polystyrene. Propane is already in the sugar before expansion, so when we got sent a box of 'sugar', we had to vent it for several days to remove the worst of the propane- even now though, if the lid is left on for a time, propane still builds up. By soaking the 'sugar' in varying concentrations of propane, mixed with minimal media broth upto 0.4 ml, to completely cover the 0.5g 'sugar' that will be put in each agar dish tomorrow. 0.4, 0.2, 0.1, 0.05 and 0.00ml of propane are used to soak 0.5g of polystyrene 'sugar' overnight on a shaker at 275RPM.

(14:30) After many problems again with the gel's Chris has finally been able to load the gel with 1/100 dilution the PCR reaction. Unfortunately with one of the gels the comb was to long and so the sample went strait through... meaning that we lost the majority of the first two samples. Lane organisation
1 = Aeruginosa +ve control
2 = orange 100x dilution
3 = orange 100x dilution
4 = orange 100x dilution
5 = yellow 100x dilution
6 = yellow 100x dilution
7 = yellow 100x dilution
8 = yellow 100x dilution
9 = mixed 100x dilution
10 = mixed 100x dilution
11 = mixed 100x dilution
DNA Hyperladder 5ul
12 = negative control bench
13 = negative control hood this is to be ran for 2.5 hours at 120V . each sample was 20μL apart from 1 and 2 which there were only 2μL and 5μL left respectively

(15:40) gel has been running for 1.1 hours now and the loading dye is almost at half way. Luke is now about to load the bacteria into the Pentane Poly experiment. Nathan is starting to do the gel extraction of the PCR reaction of the orange and yellow single 16S. primers have yet to arrive. Will had just spec'ed the experiment with the Yellow, Mixed and Orange colonies, which looks promising, however we are going to re spec, and do this experiment again in triplicate to make sure that it is not just chance, as well as the pentane with poly experiment to hopefully rule out this.