Lab work (Patrick, Anastasiya and Anja) 06/07/10

After last weeks succesful ligation of CAM and KAN resistance markers into plasmid pSLD3, we successfully constructed plasmids pSLD30 (which contains CAM) and pSLD31 (which contains KAN). All the steps and how we calculated the volumes needed of all the items for both restriction and ligation can be found in the lab book. Long story short, in order to have ample amounts of inserts, (FP's and resistance markers), we performed PCR's.. both de-novo amplifications of the inserts that weren't present as PCR products from Sebastien already, and re-amplications of the PCR products available from Sebastien. Once PCR was completed, we had ample amounts of all the FP's (yGFP, CFP, and CRFP) as well as resistance markers (CAM & KAN).
Next step was to perform a PCR clean up; in order to construct pSLD30 and pSLD31, for now we were only working with the resistance markers.. FP's will come in the picture at a later stage. So, a PCR clean up was performed (follow protocol) on the PCR products of CAM and KAN. These are now ready to use for the next step, which were restrictions.
The primers used when performing PCR on CAM and KAN had tails which contained restriction sites, which brings us to the point using restriction enzymes. The table with all the combinations of restriction enzymes used for each digestion can be found in the lab book, the point was to perform restrictions such that in the next step we could ligate plasmid with insert in the combination of:

• pSLD3 + CAM = pSLD30
• pSLD3 + KAN = pSLD31

• PCR
• PCR clean-up
• Restrictions
• Gel-control of restrictions
• Ligation
• Gel-control of ligation
• Transformation
• Plating
• Over-night cultures
• Plasmid purification & -80 freezing cultures

Lab work (Thomas, Anja, Patrick and Maya) 25/06/10

Today Thomas joined the lab:-)
The 10-1 and 10-2 dilution from the 24/06/10 yielded several hundred colonies which means that the transformation was indeed alright. As it's Friday today, we will wait with the O/N culture till Sunday.
The O/N of strain SP58 showed growth this time. The plasmid was purified and stored together with the FP plasmids. A -80 freezing culture of this strain was also made.To get an overview of the strains and plasmids we have used so far, have a look in the strain and plasmid bank in dropbox-research group-strain and plasmid bank.

You can't spell funding without fun... (Annemi) - 25/06/10

I had a look at some funding and where we should go from here.

• We should definitely apply for the COWI fund. This will take some time since they have a lot of requirements for applying. (see http://www.cowifonden.dk/Vejledning_for_ansoegere.asp)
• The ‘Fondeliste’ has been updated according to the funds that Anastasiya and Anja found in the beginning of June.
• A document called ‘Company applications’ can be found in the ‘company’ folder. Please write in that document if you send out a company application.
• An application to the fund ALECTIA has been send.

Birthday and fun day at the lab? (Maya, Anja, and Patrick) - 24/06/10

So today was yet another day in the lab, only difference being it was my birthday (Patrick). :D
The serial dillutions we plated of our transformants didnt seem to produce a substantial amount of colonies after having plated them yesterday, so we plated a 10-1 and a 10-2 dilution to get a higher number of colonies.
We purified plasmids from strain SP44, SP45 and SP46 using the miniprep kit, and ran the purified plasmids on a gel to check if our plasmid purification was efficient and successful. There is now a miniprep protocol in the protocol folder.
We also made some -80 freezing cultures of strain SP44-46. To keep track of the future freezing cultures we have made a document called strain bank which we should all use when we make some -80 cultures. It's in the research group-> strain and plasmid folder.
The O/N of SP58 didn't show any growth, so a new O/N was started.

Progress (Maya, Anja, and Patrick) - 23/06/10

So we started in the lab yesterday after having gotten all the needed information from Flemming concerning the FP's (fluorescence proteins).
We started constructing some template plasmids with FP's and markers, and so far its been a fun experience thanks to Hassan.
As things are now we have decided to use the fluorescent proteins mCherry and YGFP as the reporters, though the final template plasmids will include the fast-degradable fluorescent proteins of mCherry and YGFP.
Today we started O/N cultures of the following strains: SP44, SP45, SP46 and SP58. SP44-46 contains plasmids with CFP, yGFP and Cherry, and SP58 contains a plasmid with the markers CAM and KAN.
We also made a transformation using electroporation. We transformed plasmid pSLD3 into E.coli strain . pSLD3 is to be used as a recipient plasmid of markers and FPs. After the transformation, we made a serial dilution of the transformed strain and plated these. We have uploaded a transformation protocol in the research group folder- protocol folder.

iGEM the last 2 weeks - 21/06/10

So the last 2 weeks have been quite hectic and busy, and if you're wondering why there hasn't been an update its because alot of things keep coming up while working on the design.
The in-silico design is moving along well, though when every new step comes along we run into issues, just like today we realized that the UV system just might not work after all, long story short - because the excitation wavelength will interfere with the excitation wavelength of the red-light receptor. Solution: good old lov-tap will most likely come into the picture now.
In terms of the reporter genes, we were lucky enough to find Flemming back at CSM today, and so Maya and Juliet managed to get alot of good info from him about flourescence proteins which made us wiser in that area, although we also realized they arnt as straight forward to work with as initially thought, so we need to put in more work in that area as well.
We are set to get into the lab tomorrow to possibly start construction of the template plasmids with the flourescence proteins, that is of course if we have the FP's we want to work with and whether those have the appropriate degradation time.
That's about all for now, stay tuned for more exciting news in the days to come.

Maya, Anja, Malthe and Patrick's log - 08/06/10

We have finished the step-wise construction of our system, and have begun on the in-silico model.
We have uploaded the initial work from the in-silico model in a newly created folder in the research group called step-wise construction and in-silico model.

Maya and Patrick's log - 07/06/10

So today we have been working on the step-wise construction of our system, taking into account how to test each part along the way. We learned a lot about recombineering and the factors entailed in the process and have come up with the initial version of how we are going to construct the system.
As a rule of thumb we also learnt the following combinations and bad-combinations when working with FP's:

• RFP can be used together with either GFP or CFP; and YFP together with CFP.
• The bad combinations which we should never ever do are the following: RFP with YFP; and YFP with GFP.

Captain's log 040610

Maya, Thomas, Patrick, Lisa and Annemi have been working on a project description for Sebastien. The document 'project description' can be found in dropbox in the research group folder. From now on this document will be the only one describing the project, therefore you should edit in the document and not create a new one. We have decided that every time we are doing something iGEM related, that it should be posted in this document. Please write the date and your name at the beginning of your log. This will make it easier for all of us to keep track of what is going on in the group. We have tried to make a simple plan of what needs to be done before going to the lab:

• Investigate the E. coli chromosome to find out where we can integrate the switch
• Find the BioBricks we will be using (DNA sequences)
• Install the software CLC main workbench (the program which will be useful for the design of the system)
• Design the system: Order of genes, restriction sites, DNA sequences, order of integration of genes into the plasmids
• Which plasmids (Sebastien)
• Primer design
• How to test that the genes have been inserted (antibiotic resistance)
• Make a phase planning and talk to Peter (divide into smaller parts and and test these individually)

Maya will start creating an illustration for the system during the weekend. Thomas will delete all irrelevant budgets from the dropbox. He will also send the project description to Sebastien.