Diary of the TU Delft team

11th September, 2009

General Today we had a video conference meeting along with Calin from Sweden through skype. We asked everybody's opinion on whether Esengul can come instead of Orr to Jamboree. Everybody accepted and appreciated the decision. Since our team is allowed to attend only one and half hours of the Kluyver centre's meeting and also there is a huge amount of lab work pending we decided to make sure can only Daniel and Saeed attend the meeting. We will decide after discussing with other Holland-iGEM teams. Calin He is updating the modelling of the delay module in the wiki. He also needs to do more modelling and he was suggested that Domenico and Emrah are willing to help any of the issue he faces in modelling. Tim Vos He has got 172 reponses for the ethics survey. The deadline for ending the poll was set as 21st September. He also decided to send a mail around to all iGEM teams about ending the poll. He is also updating the wiki for the ethics and he is also going to sought the help of Suzanne of Biotechnology and Society who has also shown willingness for updating the wiki. Weenink He has assured that the final assemblies for self destructing module will be completed next week. He is also planning to test the enzyme's role of cutting the plasmid. He also discussed with Calin about the knock out kits. He is also going to test why the sequencing is getting failed, by doing PCR using the primers given for sequencing. Saeed He is going to meet the Delft Gementee for sponsorship next week. He is also planning to interview our Biosafety officer Lesley Robertson for the microethics. He is planning to finish the communication plan and send it around to us. Sriram The assembly of the LacI generator is getting over today. It has to be attached to the other final assemblies and tested next week. The other sequencing opportunities must be checked and finalised. He is also planning to do the experiments with plate reader to test the delay constructs.

4th September, 2009

General Today we had an elaborate meeting on many issues. All our supervisors Janine, Emrah and Domenico were present. Calin was not able to attend the meeting through skype and Orr also was not able to attend the meeting. With others we conducted a poll to choose the top 3 categories in which our project is most applicable to. The results are 1. Information Processing, 2. Foundational advance 3. Health and Medicine.

The deadline for the decision of attending the Jamboree for Emrah, Domenico and Janine was set as 10th september so that flight tickets could be booked. It was also decided that Saeed will present about our project in the Kluyver Centre meeting in Wageningen. In the meantime it is also decided to choose who will do the presentation in Jamboree, by next friday's meeting 11th september. Saeed and Tim Vos had volunteered for speaking about the sponsorship with Gementee, Delft. Also Domenico and Emrah has shown their willing to help in the lab works. Everybody appreciated the new wiki page.

We discussed on the topic of "how much of our project must be communicated to public?" for the microethics. Many agreed that the public must be informed fully about the project except some sensitive details that could be misinterpreted by them.

Tim Weenink The lab work is progressing well, he prepared k12 electro-competent cells and will know the results by monday. If those strains work well the selfdestructive plasmid will be finished in 2 weeks. With the help of Calin he needs to look into some knockout kits so that knockouts can be continued for conjugation module.

Sriram He has started updating the lab notebook. The final assemblies of locks and keys are getting over by monday. If the K12 strains work well then the experiments can be started by wednesday. There were some problems in sequencing results from Baseclear. To find whether is there some problems with primer, PCR must be done on monday with the primers. He got some comments for the new wiki page made by Calin. Vos said some pages miss some words due to some misalignments with new template. The gel images posted in the lab notebook can be put as thumbnails and the table showing details of the gel with its image can be put in a new page called results, along-with links to give navigation between both lab notebook and the results page. The reference in the home page must be removed and all the images must be resized for new template. He suggested whether the color of the bacteria in logo be changed to orange as in template. Others suggested, it could be changed but after changing the decision will be made after a poll on the new color is conducted.

Saeed He will be available only on mondays and fridays since he is staring his internship with Parliament and is helping to finish the self destructive plasmid module.

Tim Vos There were 153 participants for the survey. Still now there is no reply from iGEM HQ for posting our survey link in the iGEM website. But has posted the link in iGEM forum.

Daniel He wont be available for labwork anymore since he has started his thesis this week but occasionally will check with Sriram and give suggestions for the progress of Delay module. He has given all the informations needed for Sriram to proceed with the Delay module. He is also working on the description of delay module for project overview.

27th August 2009

General Remarks: On September 25th, there is KluyverCentre Programme day in Wageningen. We have a presentation there (what? how? who?). Everybody is expected to attend. Details about car pooling, payment etc will be arranged. Some decisions on the shirts and sweaters to be worn during the jamboree is taken.. Details will follow soon, actions will be taken by Saeed.

Sriram: Started the assembly for the ribo regulator. among 3 steps to be taken, first one worked without any problem. Consecutive steps will follow soon. The university is providing a virtual environment for iGEM, that can be used for either wiki or forum or anything that is suitable. Arrangements should be done and actions will be taken by Sriram and Emrah

Daniel: LacI problem. The problem is described as:

GFP with weak RBS and controlled by PLacI. However, in the experiments i did today PLacI acts as a constitutive promoter and the culture always glow. This makes me think on either I made a mistake and put a constitutive promoter or e coli is not producing LacI. Do you think that the second reason is possible? i'm almost sure is not the first one, ha. Anyway i was looking in the registry and i couldn't find a biobrick with LacI controlled by a constitutive promoter (no PTet because that promoter is in the cascade). so we should assembly one ourselves.

As the solution. There are 4 steps to be taken:

• Sequence the promoter
• Post in iGEM forums, maybe other users are also experiencing the same problems.
• Contact the other teams who posted that they are using LacI
• Contact iGEM HQ.

Daniel will take action on those points. Worst case scenario, LacI will be removed. Furthermore, Daniel contacted some iGEM teams to ask how did they do the lock-and-key ribo-regulators. So far, 2 different lock-and-key pairs are designed, characterized (medium and low RBS) and the 3rd one is communicated with the other iGEM teams.

Lastly, one organizational note: Daniel will start his MSc project at the beginning of September.

Saeed: Self-destructive plasmid. Have done the 1st assembly (out of 3 step plan). The last assembly is expected to have the T4 ligase/endonuclease problem.

21rd August 2009

Saeed

There are concrete output(s) on the sponsoring companies/departments: e.g. DSM, Baseclear, Dept. of Nanobioscience. The logos on the wiki etc... will follow soon. There will be a a newsletter produced before the Jamboree. Collaborative (together with other Dutch universities) contact with the Dutch embassy in USA is planned.

Will act on 'Question of the week' later.

Calin

Conjugation tests are being held. Sizde exclusion tests are OK for small plasmid bu a bit problematic with the big plasmid (trb protein issue)

Daniel

Delay project: Working with cascade in parallel with the lock-key. Negative feedback cascade constructs are working fine so far.

Weenink In the first place we used BBa_K142205 which already was available in the regiostery(its a I-SceI endonuclease gene inclusive promoter and terminator etc.) Agarose gel showed this fragment is 400-500 bp larger than it should be. After sequencing it seemed that there is no Endonuclease there but a T4 DNA ligase.

BBa_K142202 is also available in the registery(it is the same as BBa_K142205 but without promoter). Tim checked this biobrick also by sequencing and it seemed that it is the right part, as I understood. But after assembling BBa_K142202 with a pLAc promoter the same results showed on agarose gel as with BBa_K142205, same fragment size. So last friday we tried to sequence this biobrick but this sequencing failed.

Otherwise things are going as smooth as planned.. Probably next week we'll have the final construct (Absent next week)

Sriram

In delay device: 1st plan did not work, luckily the backup plan did. The problem is that final microlitres of T4 DNA ligase & SpeI endonuclease were not showing best activities and hence we ordered new ones from Ginko biolabs. In the meantime we tried to use the lab enzymes from other company which obviously didn't work because of buffer conflicts.

We have to order miniprep kit.

Vos

The questionnaire is ready to be sent around, waiting final feedback from Emrah. Helping people in the lab, absent next week.

20th August 2009

We had a big meeting with Groningnen, Amsterdam and Delft

22nd July 2009

Update of procedures:

• Amount of cells grown by the delay device team is quite small
• Transformations for the delay device team did not work as well as expected; it is unknown if it is because the cells are incompetent, the transformation process went awry or too much antibiotic was used
• The delay device cascade model has been upgraded (we have 30 parameters in 9 different equations (IPTG diffusion equation, 4 ODE's for the mRNA concentrations and 4 ODE's for the protein concentrations))
• Some modelling conclusions: if there is leakage, there is no delay (if leakage is higher then 10^-5%, the delay would be less then 1 minute, where the maximum delay is 400 minutes)
• mfold hybridization software was used to hybridize the secondary structures of locks designed by Daniel and key3c designed by Berkeley 2006 iGEM team. The kinks and unpaired bases were modified by changing the key3c sequence to make new keys fitting the locks
• Self-destructive plasmid: assembly has begun and succeeded, biobrick amplification was done
• Press release: we have an article on our iGEM team that will be in the Dutch newspapers soon
• Our project has a potential application for future work on antibiotic resistance by understanding bacterial conjugation. Another potential application is in bacterial communication
• 52 teams have responded to the Macro-ethics email, and are willing to take part in our survey

To do:

• We should make a plan for the laboratory work in case things go wrong
• For the modelling, we should model parameter ratios rather then parameters
• For the lock and key secondary structures, create a program that can automatically modify the sequence of a key to fit into a lock
• Make the conjugation modelling compatible with the conjugation lab
• Try to think how to incorporate the thermodynamic output from the hybridization mfold program to into the riboregulator Matlab script

22nd July 2009

Today our meeting focused on what we can do in terms of public relations. Some suggestions which came up included updating the first page of the Wiki so it will be more understandable to laymen. One analogy which was raised to simplify our project was that of relay race, where the torch is passed from one bacteria to another. We also decided to give our project a name related to this relay race idea. In addition, we discussed possible applications which we might have, since they would also help our PR. Two applications that were raised so far were:

1. Kill bacteria with messenger bacteria by conjugation - a problem that arises with antibiotic treatment of bacterial infections in people is that bacteria build resistance to antibiotics. Using a bacteria with a messenger plasmid, it would be possible to use conjugation to send a plasmid into another 'bad' bacteria and destroy it from the inside
2. Multi-tasking bacteria with temporal control of task performance - conjugation can be used to keep communication between bacteria networks. The messenger plasmid can be used as a signal to initiate a certain task in another bacteria, and after some time the bacteria will stop performing the task (by self-destruction of the messenger plasmid)

Yours in unity, TUD iGEM '09

15th July 2009

We reported that our lab work started to move, especially for the delay device and self-destructive plasmid. We reported that the transformations were done, and now we are just waiting for the colonies to grow. In terms of modelling, we reported a successful building of an algorithm in Matlab to model the delay device, including the various parameters and equations necessary. We decided to simply continue in the same way we did so far.

Yours in unity, TUD iGEM '09

8th July 2009

Today we mainly discussed the plan that we had for the coming couple of months. We had an overall plan on when we will be doing what, and then each of the groups presented their own sub-part and what they were going to do. For the delay device, there were 4 different approaches that could be taken, and actually 2 of them were deemed useless to use, which already narrows down the options. Regarding the self-destructive plasmid, there was a design for it, but there are still some things to work out. Finally, for the conjugation, it seems that there is a pretty good idea of how it should be done.

Yours in unity, TUD iGEM '09

2nd July 2009

Oh glorious day! Oh marvellous day! We finally decided the topic we will do, and this time we are certain this is the topic: self-destructive plasmid (the title is still in the working). We also established a complete research question and a division of the work. By next meeting, we decided to come up with an overview for each of the three parts we will be doing.

Yours in unity, TUD iGEM '09

29th June 2009

Dear Diary, today we had a pleasant field trip to Groningen, which is at the North of The Netherlands (we're in the centre, so it took about 2 hours to get there). We met there the iGEM team from Groningen, which were quite a nice bunch. We had a chance to present our own ideas for a project, and they presented theirs. After seeing their labs, and how organised they were in terms in of dividing the tasks between all of them, we decided to start pushing the project forward, and by the end of the next meeting to decide on the project idea and how we are going to divide the work between us. We also had much time to talk on the way back from Groningen, when we also decided to be more united as a team, for example by doing some activities together (e.g. playing soccer, paint-balling, and so forth).

Yours in unity, TUD iGEM '09

23rd June 2009

Dear Diary, today was a very, very long meeting, where we discussed many different issues (mostly about the project, but also about acquisition of funding, travelling arrangements, meeting with Groningen etc.) We also had a vote about the different features concerning our two candidate projects: the self-destructive plasmid and bi-stability. We gave a grade of 1-5 (1 being worst, 5 being best) as our rating for features such as feasibility of the project, ability to model it, how interesting it would be to other people, how applicable it might be for further research, etc. All the results can be seen on the Ethics section. We realised that we have quite varied opinions about the different features of both ideas, which is quite interesting from an ethical perspective (how each one considers the project, mainly in terms of feasibility and acceptance by the iGEM judges, by the science community and by the general public).

Yours in unity, TUD iGEM '09

17th June 2009

Dear Diary, today was a somewhat disappointing meeting. Two members of our team worked very hard on the literary research, and the rest of us didn't really help them. We promised to ourselves that we will participate more in the research, and that we will do our best to get a finalised plan by the end of June, so we can start doing something.

Yours in unity, TUD iGEM '09

11th June 2009

Dear Diary, it was quite a disappointing day. First of all, we had a small presentation in the morning, but about half an hour before it we had a terrible realization: our idea is fundamentally wrong!!! :-(
How did that happen? Well, when we electroplate selection markers into the plasmids, expressing them will allow the host for the plasmid to pass the selection. Unfortunately, expressing them would express the entire plasmid, including the self-destructing gene, meaning that after selection the plasmid will be destroyed before we even use it. On the other hand, if we give the host a plasmid with a selection marker but don't apply any selection, the plasmid will be ejected from the host, so it will not contain the self-destructing gene.
Luckily, we came up with an alternative idea: Fucking Conjugates (literally). The essential idea is based on bacterial [http://en.wikipedia.org/wiki/Bacterial_conjugation conjugation], where an F plasmid in F+ E.coli is transferred into an F- E.coli. In an experiment done by [http://parts2.mit.edu/wiki/index.php/Berkeley2006-ConjugationMain Berkeley in iGEM 2006], they took the origin in the F plasmid (which is a signal to transfer the F plasmid from one E.coli to another) and put it in a micro F (mF) plasmid instead, so that the mF plasmid was transferred, and not the F plasmid. Our plan is to extend on this idea by applying oscillation, self-destructive gene and timer, but we are still not sure what research question it can answer. So the idea is there, but we still need to figure out the purpose. :-P

Yours in unity, TUD iGEM '09

5th June 2009

Dear Diary, did we have a long discussion today! First of all, we determined how to divide the work among us (each one said what his skills were, and we decided who will do what in the project). Obviously, some of us took more managerial positions (taking care of planning, logistics and what not), and some were more on the employer/slave side (Emreh even suggested that a whip might come in handy...)
Later, we discussed the initial steps of working with the self-destructive plasmid, which pretty quickly turned into several simultaneous conversations: whether we should use a selection marker or not (somewhat pointless, since the marker would be already given in the plasmid supplied from MIT), methylation of the plasmid nucleotides (which no one really knew how it is affected after replication of E.coli) and the rate of plasmid degradation compared to gluing them back together. In the end, we gave some tasks out: Daniel was responsible for finding the top 10 restriction sites in the E.coli plasmid, Sriram was in charge of going over the registry of the bio-bricks of iGEM to seek a useful promoter, and Orr would look into methylation in E.coli to expand more on how it works and how it is affected by replication.
In short, much to do, for all of us. LET'S GET WORKING!!! :-D

Yours in unity, TUD iGEM '09

26th May 2009

Dear Diary, today had a brainstorm about our main 6 topics again, and we realised that the most feasible idea we can choose is the self-destructive plasmids (some of us already had PR ideas, like a poster of James Bond with a gun-shaped plasmid in his hand). Finally, we agreed to write a short plan on what we will do and how each one of us shall contribute to the project.

Yours in unity, TUD iGEM '09

14th May 2009

Dear Diary, today we looked at the different ideas we had for the project. Needless to say, most of the ideas seemed more appealing as we started looking deeper into them, although it was somewhat disappointing when we found out the salt water desalination using bacteria was not quite feasible. The micro-organism muscle idea also turned out to be a flop. However, there were some nice developments in the other 4 ideas, and the buoyant bacteria application in mines seemed quite promising.

Yours in unity, TUD iGEM '09

8th May 2009

Dear Diary, as we decided in last meeting, we chose the top 5 ideas from the list of subjects we had so far. We came to the following conclusion based on democratic voting:

1. Salt water purification
2. Buoyant Bacteria
3. Impulse Signal
4. Melatonin Compensation
5. Micro-organism Muscle
6. Enzyme Modulation (shared 5th place)

We agreed to divide the subjects and look at the possibilities, opportunities, feasibility and applications for the top 5 ideas, so that next meeting we could inform each other on the subjects, and possibly narrow down the list to a top 3.

Yours in unity, TUD iGEM '09

28th April 2009

Dear diary, today we had a meeting regarding the practical aspects of the iGEM project (we divided the task of looking at the project ideas among all the group members, and decided that each person would look at 3 ideas, make a small research about them and publish it on the website, so that we can choose the top 5 ideas, and out of them choose the most attractive one). We also agreed on starting to look for financial support, but then we agreed we should wait a bit longer until we have a concrete idea for our project. We also agreed on meeting with the designer of the Delft Wiki page from last year, so we can learn from his wisdom and bask in his glory.

Yours in unity, TUD iGEM '09

23rd April 2009

Dear diary, today we had a brainstorm session, where we invited various people, including professors and experts in different fields of biology, chemistry and engineering. Sadly, only 3 people outside our team have come to our aid, yet we got some good ideas and feedback from them. We ended with 21 ideas, some of them being highly general and highly important (e.g. salt-water purification), some started as a joke (e.g. flashing dog poo brought an idea of fast degradation of dog excrements) and some were simply cool (e.g. flashing bacteria colony). In the end, we did not decide which project we shall do, but we decided to look at all the projects ideas and look at the feasibility, advantages and disadvantages of each idea.

Yours in unity, TUD iGEM '09

17th April 2009

Dear diary, today we continued with looking over past projects. We also discussed the possible sponsors we might be able to recruit, including the town of Delft (by advertising them on the Jamboree as a town worth visiting) and various beer companies (specifically in case we would make a project involving beer). We also discussed the Teacher workshop in London (30th-31st of May), and since only one student can accompany the instructors, we decided to hold a fight among all students who would like to go, and last one standing would go (if this would not work, we'll just make a lottery). We also agreed to have a brainstorm regarding the project we will choose on the 23rd of April, where we will invite people to come and give remarks on our ideas after Daniel would give a short introduction of iGEM and our project ideas. Hopefully, enough people will come to make it worthwhile.

Yours in unity, TUD iGEM '09

7th April 2009

Dear diary, today we gave some presentations about iGEM projects of previous years. Some of them were pretty good (the Slovenian team of 2006 came up with the idea of making a sepsis-free cells, and the 2007 Slovenian team came up with ubiqiotinating methods against HIV infected cells) and some were just highly extensive with little concrete results (the 2008 Cambridge team attempt at making an artificial neuronal network for E.coli was not quite a success...) We also saw some 'cool' experiments (gladiator bacteria and generation marking in bacteria), but we still haven't found the project we want to work on. Nevertheless, we had some excitement about a project by a TU Delft Masters student working on a bacteria that can sense whether it is in an environment with low insulin concentration and produce insulin in response (could be a good diabetes treatment).

Yours in unity, TUD iGEM '09

25th March 2009

Dear diary, today we went over some general points, regarding the booking of the hotel in Boston and how we can take an extended vacation in the US after going to the Jamboree.

Yours in unity, TUD iGEM '09