How the collaboration was 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 feedback 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 has 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 estrogen level in water.
The second project focuses on a biological system that utilizes cytochrome P450 79's from plants to produce small molecules called oxides, 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 Standard Assembly of BioBricks.
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 in this system there is no need to eliminate restriction sites. 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.
Our initial idea was to construct a plasmid from scratch with both the Plug 'n' Play with DNA and the Standard Assembly of BioBricks, so we could compare the two systems. Due to our late entry into the iGEM competition 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 criticism as well as testing each others wiki's.
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 with DNA standard.
When more than one illegal restriction site is present at a certain distance of each other, more than one round of site-directed mutagenesis by PCR has to be performed. A circular arrow in the figure below illustrates this.
Work conducted for The Copenhagen Team
We in total created seven BioBricks for the Copenhagen team; these parts and our standard backbone plasmid were intended for assembly four different plasmids. Two with human the human CYP's A2 and C9 as well as two with the plant CYP's 79-A1 and 79-B1. All pre-produced parts for the assembly were delivered to the Copenhagen team for them to assemble according to the Plug 'n' Play standard. The two plant CYP's were assembled without problems. The human CYP project was however put on halt. The two constructed plasmids with CYP79-A2 BBa_K527001 and CYP79-B1 BBa_K527002 are illustrated below.
Plasmid with CYP79-A2 BBa_K527001:
Plasmid with Cyp79-B1 BBa_K527002:
The plasmid with CYP79-B1 BBa_K527002 could in spite of repeated attempts not be assembled with the Standard Assembly of BioBricks. As mentioned, it was however possible to assemble the plasmid with CYP79-B1 BBa_K527002 with the Plug 'n' Play assembly standard. The plasmid was proven to work in accordance with the control. The Copenhagen team managed to extract the cytochrome membrane protein, verify it by western blotting and finally show that the cytochromes could produced oxides, with the help of our assembly standard.
CYP79-A2 BBa_K527001 was assembled with the Standard Assembly of BioBricks, but unfortunately the CYP79-A2 BBa_K527001 was never proven to work in accordance with the control. The lack of function could have been caused by the point mutations that were introduced in order to remove the illegal restriction sites.
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