How the collaboration formed
In the beginning of July, we arranged a social event with the danish iGEM teams; Copenhagen and DTU-Denmark. The Technical University of Denmark and the University of Copenhagen are geographically located close to each other. At this event each team presented the outline of their project and afterwards there was set of time for feed-back and discussions. The gathering was very rewarding and productive, and it resulted in a good collaboration between our team and the team from Copenhagen.
The project idea of The Copenhagen Team
The Copenhagen team have two interesting and ambitious project ideas.
One project focuses on removing pollutants derived from pharmaceuticals and personal care products from water. The idea is to introduce different types of membrane bound human cytochrome P450's (CYP) into E. coli and following investigate if the cytochromes have an impeding effect on the oestrogen level in water.
The second project focuses on a biological system that utilizes cytochrome P450 79's from plants to produce small molecules called oximes which inhibit mitochondrial peroxidases in fungi. The system is intended to be introduced into E. coli which would then kill the fungi because they would not be able to break down the hydrogen peroxides.
The collaboration
The foundation of the collaboration between us and Copenhagen was the DNA sequences of their CYP's.
As is often the case with eukaryotic gene sequences, the CYP sequences from humans and plants contained between one and eight illegal restriction sites according to the BioBrick standard.
There was put further time pressure on the team as they experienced problems with acquiring sequence information of the human CYP's, which delayed the project. Furthermore the elimination of the illegal restriction sites by site-directed mutagenesis caused a lot of trouble because of the number and positions of the restriction sites. This can in general be a big problem if you are under time pressure or have limited experience – especially with DNA sequences originating from plants, fungi and mammalian cells.
When we met mid-July, Copenhagen had spent a lot of time trying mutate the illegal restriction sites, and they still had to eliminate a lot more.
Fortunately, the assembly standard that we have designed is easily adapted to any molecular biology research project, and the in this system no restriction sites are illegal. By using our system they could save a lot of time, and that would allow them to proceed with the more interesting part of their project. This was also a great opportunity for us to test, if our system really could be customized and applied to a random research project. It was an opportunity to test our assembly system and the expression of the resulting plasmids in E. coli.
An initial idea of our project was to perform both Plug 'n' Play with DNA and the Standard Assembly of BioBricks simultaneously, so we could compare advantages and disadvantages of the two systems. However, we did not have time to do it ourselves, but the Copenhagen team tried out both systems and compared them.
Another part of our collaboration has been practicing our presentations and giving each other constructive critics as well as testing each other wiki.
The figure below illustrates the process that the Copenhagen team had to go trough with the Standard Assembly of BioBricks compared to assembly with our Plug 'n' Play assembly system based on USER cloning.
When more than one illegal restriction site is present, more than one site directed mutation by PCR have to be performed, which in the figure is illustrated by a circular arrow.
Conducted work done for The Copenhagen Team
For the Copenhagen team we in total created BioBricks for assembly four plasmids with two human p450, p450-A2 and p450-C9 as well as two plant cytochrome p450, the Cyp79-A1 and Cyp79-B1. All pre-produced part for the assembly was delivered to the Copenhagen team for them to perform the Plug 'n' Play assembly by them self. The two plant Cyp79 was assembled without problems. However, the human Cyp project was put on halt. The two constructed plant Cyp79-A2 BBa_K527001 and Cyp79-B1 BBa_K527002 is illustrated below.
Device with Cyp79-A2 BBa_K527001:
Device with Cyp79-B1 BBa_K527002:
The Cyp79-B1 BBa_K527002 was never accomplished to assembled by Standard Assembly of BioBrick. However, the Cyp79-B1 BBa_K527002 was assembled with the Plug 'n' Play assembly system and proved to work in accordance with the control. The Copenhagen team manage to extract the cytochrome membrane protein, verify it by western blotting and finally showed that the cytochromes could produced oximes.
Cyp79-A2 BBa_K527001 was accomplished to assembled by Standard Assembly of BioBrick. However, the Cyp79-A2 BBa_K527001 was never proved to work in accordance with the control. The lack of function can be due to alteration cause by the point mutation.
|
"
