Team:Groningen/Notebook

Starting up
In March we started with brainstorms once a week. During these metings we decided on a project. We had a lot of different ideas. In May we decided to focus on three of the ideas we had:
 * Using flagellar rotation of Bacteria to make a pump of liquid inside a tube.
 * Making E. coli have a memory of events, like light inputs.
 * Make a cellular automata in E. coli for instance Conway`s game of life.

The first idea was hard to make in the 2 or 3 months we had. Here we needed to have bacteria attach to a surface and also have the same orientation in order to have the movement of liquid in the same direction. This topic would probably take us longer than 3 months, so we decided not to move on with it.

For the memory idea we wanted the E. coli insert a small piece of DNA into a plasmid after it got an input. There is a phage system which put an insert inside a certain code in the DNA. However the problem is that we need bacteria to have all just 1 insert at the same time. Moreover we wanted more than one input behind eachother and the bacteria getting inputs in the same order inside a plasmid. This last is probably really hard to obtain, and not possible in the amount of time we have.

The last idea, making a cellular automata seemed to be our best option, since we really like to make a system inside E. coli and have a nice object to model.

The labwork
The parts

In June we started to transform E. coli's with the parts. We used the strain DH5alpha, since these we already present at the university. However, we obtained hardly any colony's. We solved this problem by using Invitrogen's Top10 competent cells and making Top10 cells competent ourselves. Still we didn't have a lot of colony's, but we obtained quite some parts. The last parts we really needed for the system, were requested from iGEM HQ.

Cloning the parts together

Parts were assembled according to the Standard Assembly from the parts registry site. When we had to assemble a small part to a big part, the big part was ligated into the vector containing the small part.

Media

For testing how HSL could diffuse trough the media we wanted to use two parts, BBa_F1610 as sender device and BBa_T9002 as receiver device. Unfortunately the sender device from the registry seems to be wrong (Restriction / PCR /Sequence analysis confirms this), therefor we used the sender device from the Ron Weiss group. In TY browth medium we observed really high GFP background from part BBa_T9002 even without induction. By using EZ rich defined medium the background was reduced. For further measurements we decided to use EZ medium.