14-09-2010 Anja (Repressor group)

Lab stuff:

• Annemi minipreped the overnight BB transformation cultures from yesterday. She minipreped only half of the O/N, because Malthe and Annemi think we have enough plasmid to work with (Correct?).
• those minipreps labeled with white stickers are stored in the -20 degree freezer in a box with date 14-09-2010
• A back-up preparation of the transformants has also been made. 2x2mL of cells have been centrifuged and the cell residue is kept in the -20 degree freezer (white stickers: date, biobrick/plasmid #, 'cells') This will make it easier to prepare a new mini-prep since we don't have to grow overnight cultures first.
• a gel of minipreps has been run and I took a picture. Seems like the mini-preps went well since a band is visible in all of the wells.
• Glycerol stocks of transformant cells have been done and stored in the -80 degree freezer
• OBS they reorganized the freezer > look into lab book "freezer box location"
• Malthe has resuspended our primers, which arrived today :-)
• One primer is still missing, Malthe wrote an email to ask for primer IGT02

• Look for a big 2 L box in the fume hood in our lab next to the sterile bank. It´s refilled by Bjarne.
• Go downstairs to room 162 on the 2th floor with a min. 1L bottle. The room is on the right side, go to opposite room, get keys and book from cupboard right to the door. Write in the book, how much you have taken.

15. sep. 2010 Annemi

Today we have run PCR´s to amplify the promoter region and the repressor region. We used 2µL of Sebastiens Salmonella DNA number 143 as template. (See the lab book for further detail on the procedure)
The program used to amplify the small fragments (ca. 100 bp) are called IGEM TD 100BP
For the larger fragments we used Sebastiens program: SebFC
The products are run on a gel (3-2-1)
We made transformations with the plasmids pSLD20 and pSLD22 in the DH5α. The cells are plated out on AMP-plates (20µL and 200µL)

13-09-2010 Maya (repressor group)

Today me and Anastasiya did O/N cultures of the biobricks that we transformed last week. Tomorrow we need to do a miniprep on all of these plasmids, and also prepare some glycerol stocks. We made double of the O/N so there should be enough if you want each group to have their own sets of purify plasmids.
If the primers arrive tomorrow you should also resuspend them, organize them in a "repressor group" box and make dilutions.
I've started on another A3-sheet describing the construction of the three different setups in parallel. Maybe you can continue on that or improve it. The purpose of the sheet is to make it easier for us to see which things should be run in parallel. In that way we can do all the PCRs at the same time and so on.
If you have time Lisa, you can continue on the CLC files. I'll try to finish mine today.
I also think we should find out who is going to write what on the wiki, as I think our group should write about the gifsy1 and gifsy2 promoters, the repressors and the antirepressors.

080910 Maya and Annemi (Repressor group)

We now have a strategy on how to do the labwork in the repressor group. We will need to construct six different plasmids:
pIGR01 (GF1) /pIGR02 (GF2): A pSB4A5 backbone plasmid with the two promoters pRM and pR. Downstream of the pR-promoter we will have a GFP.
pIGR03 (GF1) / pIGR04 (GF2): A pSB4A5 backbone plasmid with the two promoters pRM and pR. Downstream of the pR-promoter is a GFP. Downstream of the pRM-promoter is the gogR and gtgR respectively.
pIGR05 (GF1) / pIGR06 (GF2): A pSB2K3 backbone plasmid with a pBAD/araC promoter. Downstream of the promoter region the anti-repressor antO/antT (GF1 and GF2 respectively) is located. As a reporter the BioBrick RFP (E1010) which is located downstream of the anti-repressor.
The plasmids will be transformed into competent cells and fluorescence will be measured with the BioLector. All of the plasmids will be transformed separately along with the following combinations:
pIGR01/pIGR02 Expected outcome: GFP will be detected
pIGR03/pIGR04 Expected outcome: GFP will not be detected
pIGR05/pIGR06 Expected outcome: RFP will be detected
pIGR05 + pIGR03 Expected outcome: Both GFP and RFP will be detected
pIGR06 + pIGR04 Expected outcome: Both GFP and RFP will be detected
pIGR05 + pIGR01 Expected outcome: Both GFP and RFP will be detected
pIGR06 + pIGR02 Expected outcome: Both GFP and RFP will be detected
As a control we will use a pSB4A5 backbone plasmid only containing GFP.

• Overlapping primers which we will use to amplify the two promoters pR and pRM as a BioBrick have been designed.
• The primers to amplify the gogR/gtgR and the promoters from the Salmonella chromosome as a BioBrick have been designed.
• Primers to amplify pBAD/araC and antT/antO from the pSLD20/22 plasmid as a BioBrick have been designed.
• Primers to amplify GFP still need to be designed. How about the RBS and terminator?

31-08-2010

IGEM UPDATE: 31-08-2010 (copied to log) The Result of the Day, and what we talked about. BB REGISTRATION - REPRESSOR & ANTI-TERMINATOR

• Each research group register the biobricks needed and in our excel sheet note when the file have been added to the registry, becouse then no further editing should be done in the excel file.
  - see "our biobricks list and sandbox.doc" in Research group.
• The needed description is written in the excel sheet - see "our biobricks list and sandbox.doc" in Research group.
  - Enter a long description of the part so that users of your part know what it is, what it does, and how to use it in their projects.
  - Enter the source of this part. For example, does it come from some genomic sequence?
  - Enter any design considerations you had to deal with during the detailed design of the sequence
  - After which we have to enter the sequence and add feature annotations.
  

• Group picture
• Wiki presentation, frame set up by Juliet. presentation of what to be filled out.
• tech-track

• Go to our team page, and look at our team roster make sure your information is typed in correct. this will be on your certificat for attendign iGEM.
• Come up with 3 good applications for our stable "real" bistable switch system, as we need to select track on friday.. (or think about it) we collect them friday, and put them on the wiki.
  AREAS:
  
  • Final Tracks
  • New Application
  • Food/Energy
  • Foundational Advance
  • Health/Medicine
  • Environment
  • Manufacturing
  • Information Processing
  • Software Tools
• tech-track

• Maya and Malthe have made an improved Gant Diagram (again) take a look at it and feel free to add to it.
  • 31_08_V001_MMB_MFK_OVERALL TIME PLAN.xls in the management folder.

24-08-2010

So the competent cells we made last week have been infected by phages!!! Oh no, this is really bad cuz it's difficult to get rid of phage contamination. Sebastien thinks that it might be a T1 phage. So we are preparing to make a new batch of competent cells. This will be done Friday. All the pipettes have been cleaned thoroughly to get rid of potential contamination.
Still working on the biobrick design. We are reading a lot about biobrick standard measurements, and we have found a lot of interesting papers. The design isn't as easy as initially thought if you want to get some data corresponding to the iGEM standard.
Maya

20-08-2010 (Patrick , Malthe , Anja, Juliet and Maya)

To make it easier for everyone to follow the project I'll try to give an update on what has been going on this week.
First of all we have had two meetings, one group meeting and one meeting with the supervisors. Summaries of the meetings can be found in dropbox in the management folder.
We have spent a lot of time on getting an overview of the project, and we have tried to make a phase diagram of what should be done for next two months.
Juliet got the Wiki up and running and is trying to work out how we can do the modeling with fictional data. She is also having a meeting today with Steen to get an overview of the economy. Remember to let her know whenever you spend some money from our account!!
The characterization of biobricks have a really high priority at the moment. So mainly Malthe and me (Maya) are trying to come up with several stragtegies on how to do this and which biobricks we should focus on. We hope that by next week we have a long list with some thought out projects. The idea is to distribute the projects among the lab group so that each person/group of two gets his/her/their own project. The responsible persons should then finish the design and start on the lab work. Some of the mini projects are most likely not going to work, so that's why it's a good idea to have several.
We have also come up with a new naming system for all our parts as this is really needed when you work with the parts in the lab. This list can be seen in the research group folder-> "our biobrick list" All the parts found in the list should be registered in our "sandbox" in the partsregistry. When we start working on our biobricks, all information should be typed directly into the sandbox. When characterizing a BB Biofab, drew Andy's company have made a data sheet see our wiki

• https://2010.igem.org/Team:DTU-Denmark/Project#characterizing_our_Biobricks

Whats going on in the lab (12/08/10) (Annemi, Patrick)

So if you have done your homework and read yesterdays log ;), you would have a better understanding and update of whats going on with the project. Heres whats actually going on in the lab right now:

• From the power-point presentation "Bistable switch design - Biobrick approach" , if you have a look at slides 6 and 9 this will illustrate what we are trying to put together.
• We are digesting and ligating by the method of biobrick assembly standard as was mentioned yesterday.
• We are digesting and ligating by the method of biobrick assembly standard as was mentioned yesterday.
• Initially we ran into problems with the restriction enzymes (FastDigest enzymes from Fermentas), after a third attempt and playing around with the digestion mix, we got them to work.
• We are hoping to be successful in our ligations, which we will be transforming today (fingers crossed for tomorrows outcome ;)), the plasmid backbone used for the ligation is pSB1T3, a high-copy number plasmid that has the Tetracycline antibiotic resistance.
• Plates containing tetracycline need to be used within 2 weeks after being made as the tetracycline antibiotic is unstable. If you have to make some of these plates as we have to, ask Lisa for the protocol for making plates. You can also ask Bjarne, the lab technician in our lab for assistance. After you have gone through the laborious procedure of making your LB solution, you need to add the antibiotic (the solution can be max 60C otherwise the antibiotic will die).
• Today the tetracycline plates made will have a concentration of 10 gamma.

Lab work - follow up (11/08/10) (Lisa, Annemi, Thomas, Anja, Anastasiya & Patrick)

As the log was supposed to be updated according to the successes based upon construction of a template plasmids containing the FP's, the reason why this never occured is because it never worked and a huge change in the project occured.
After having performed the same procedures as was mentioned in the previous log entry, the colony PCR results indicated a zero success rate at constructing the FP template plasmids.
The new biobrick approach (Annemi, Patrick, Thomas, Lisa) :
A new biobrick approach was engineered to put our whole system together. We scratched the idea of using red swap to insert our half switches into the chromosome as these were not biobrick friendly..
Instead we came up with the idea of having "mini-genes" synthesized and thereafter assembled together in the manner of the biobrick assembly standard. This also means that we can in the end submit all of our pieces as biobricks.
All the mini-genes and corresponding primers have already been ordered and we're still awaiting the arival of the mini-genes.
Dr. Thomas has created a powerpoint presentation illustrating how these mini-genes are to be assembled and how everything will look like in the end.
The powerpoint presentation, called "Bistable switch design - Biobrick approach", can be found in the "Step-wise construction and in-silico model" folder in dropbox.
As you all should know, the biobrick assembly standard uses 4 restriction sites, 2 in the prefix: EcoRI (E) & XbaI (X) and 2 in the suffix: SpeI (S) & PstI (P).
Whether having to assemble biobricks taken directly from the dna distribution kit or the mini-genes, the concept would be the same and you need to follow the assembly standard.
If you need to refresh your remember about this procedure, have a look at the file "BioBrick_Assembly_Manual" which can be found in the "Research Group" folder in dropbox.
As we are under quite a bit of pressure to construct our system, the positive side of this approach is that it should be rather easy to assemble everything as long as a lot of thought is put into the digestions and ligations. ;)
In terms of actually activating the system, to start with we won't be using the light-receptor mechanisms, instead we will initially test / activate the system based upon carbohydrate sources, namely rhamnose and arabinose.

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.