How the collaboration was formed
In the beginning of July, we arranged a social event with the other 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 followed by feedback and discussions. The gathering was very rewarding and productive, and it resulted in an excellent collaboration with the team from Copenhagen University.
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 oximes, which inhibit mitochondrial peroxidases in fungi. The system is intended to be introduced into E. coli, which would be able to inhibit the capability of the fungi to break down the hydrogen peroxides. Thus E. coli would be able to kill the fungi.
The collaboration
As is often the case with eukaryotic gene sequences, the CYP sequences from humans and plants contained numerous illegal restriction sites according to the Standard Assembly of BioBricks.
This is generally a big problem when working with DNA sequences originating from plants, fungi and mammalian cells. The Copenhagen team had some problems with acquiring sequence information of the human CYP's, which unfortunately delayed their cool 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. 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 would be able to save some time, which would allow them to proceed with other parts of their project. This was a great opportunity for us to test, if our system really could be customized and applied to a random research project. It was also a fine opportunity to test our assembly system and the expression of the Plug 'n' Play plasmids in bacteria.
In the beginning of the project we planned 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 compare the two system ourselves. However, the Copenhagen team tried out both systems and compared them for us. Another part of our collaboration has been practicing our presentations and giving each other constructive criticism as well as testing each others wiki pages.
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 possible to assemble the plasmid with CYP79-B1 BBa_K527002 with the Plug 'n' Play assembly standard. The plasmid was proven to work by comparison with a control. The Copenhagen team managed to extract the cytochrome membrane protein, verify it by western blotting and finally show that the cytochromes could produce oximes in accordance with expected. Our assembly standard therefore prove to work excellent in bacteria.
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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