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Team:Heidelberg/Delftibactin
From 2013.igem.org
Project
Notebook
Human Practice
Delftibactin. Recycling Gold from Electronic Waste.
Since the Daci-Cluster genes of D. acidovorans, which are considered to be crucial for the production of delftibactin consist of more than 58 kb, we had to think of a method capable of trasferring this great amount of genetic information to our host organism E. coli. Being confronted with this task, we considered the usage of so called "Bacterial Artificial Chromosomes (BACs)" or "Cosmids. But finally, we decided to face the challege by splitting up the crucial genes of the cluster into convetional plasmids. One containing the gigantic gene "DelH", which comprises 18 kb and another containing all residual parts, herein referred to as "DelRest". Additionally, we planed to integrate D. acidovorans's methyl-malonyl-CoA-pathway, which is a crucial co-factor for one of the PKS domains and hence, for succesful delftibactin production. Because we were unfortunately not able to genomically integrate the pathway after several attempts, we decided to create a third plasmid not only containing the pathway, but as well a permeability device (BBa_...) for enabeling our bacteria to secrete the desired product. These three plasmids were constructed in different subgroups and therefore have separated labjournals. Follow the links below to read more!
Del H
The largest part of the pathway was amplified in subdivided fragments and subsequently assembled to a 22 kb plasmid, which was later co-transformed with the other plasmids.
Del Rest
"DelRest" consists of the genes DelA, B, C, D, E, F, G, L, O ad P, which encode NRPS- as well as PKS (polyketide synthethase) modules. We divided these genes on the cluster into certain fragments, which were amplified with Gibson-overlaps for subsequent assembly.
Methylmalonyl-CoA Pathway
After our attempts on trying to genomically integrate this pathway into E. coli didn't work out well, we created a third plasmid, on which we wanted to integrate it. Additionally we therafter built in a permeability device from the parts-registry for delftibactin secretion into the medium.
Methylmalonyl-CoA Pathway
After our attempts on trying to genomically integrate this pathway into E. coli didn't work out well, we created a third plasmid, on which we wanted to integrate it. Additionally we therafter built in a permeability device from the parts-registry for delftibactin secretion into the medium.
