Team:Leicester/September2012

(09:00 am) The group returned to labs in the hope that the growth curve had gone right this time. However as per usual there was too much growth over the whole weekend. Some colonies could be counted but the group decided to wait for the rest of the team to return from their homes after going home for the weekend.

(09:15 am) The group worked on the wiki writing the past weeks work in, making sure all the details were correct and in the right order.

(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. As well as that we are going to have to remake the gel as the one will made has a fair few imperfections just to be on the safe side

(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 2ul of each of our sample, and 4ul of sample number 2 which has a lower ng/ul reading. As we have only a finite amount of DNA we are to vary the amount of the markers for different conc 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 4ul
2 (4ul)
3 (2ul)
4 (2ul)
marker 2ul
5 (2ul)
6 (2ul)
marker 1ul
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 minor 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 allowing us to work out how much to use for the sequencing to be done tomorrow.

(09:00) The group running the growth curve experiment had a single important task to do this morning. That being to plate out 2 colonies on a plate each to see if they are 2 forms of the same bacteria or simply 2 separate species of bacteria. 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 based stuff before checking the results of the plates in the late afternoon.

(9:00) Chris and Luke arrived at the lab and started to discuss the pathways for the project, we have 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 get a biobrick sorted. We are looking at the TodX TodC1&2 TobA&B genes and the other ones 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 and so far have yet to find proteins that are in p.Aeruginosa.... fingers crossed we can get hold of some of the Pseudomonas putida F1 which 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 direction, and the reverse direction (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 which is good news!

Nathan editing the wiki once more.

(14:00) had 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 have been prepared ready for plating out some colonies from CSE kits onto, to see whether we can find more positive results 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 starins 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 for hopefully setting 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 270ul of the master mix which is enough for aliquoting 18ul into 15 reactions. reaction mixture list:
171ul PCR H20
60ul HF buffer
6ul dNTP's
15ul Primer A 28f AAGAGTTTGATCCTGGCTCAGA
15ul Primer B 519R GWATTACCGCGGCKGCTG
Template DNA to e added later as it is not PCR clean
3ul DNA Pol
These reagents were added then as the DNA Pol was in glycerol the tube was mixed and span for a second to remove bubbles. dNTPs cannot be freeze thawed so whatever was left was thrown. After this, in the hood Chris took 18ul 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 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)

(9:00) Chris, Will and Luke turned up, Luke is righting 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.

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

( 11:16) Chris has now loaded the and is it running 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
5ul Marker 100bp thermo scientific
13 -ve Bench H20
14 -ve Hood PCR H20
after realising when loading the gel Chris hadn't left room for a marker with the dilutions, the smallest dilution of the yellow colony was removed as the yellow was a lot less dense to begin with, with the