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Team:Heidelberg/Delftibactin
From 2013.igem.org
Project
Notebook
Human Practice
Delftibactin. Recycling Gold from Electronic Waste.
Since the Del Cluster genes of D. acidovorans, which are considered to be crucial for the production of delftibactin consist of more than 58 kbp, we had to think of a method capable of trasferring this huge 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 kbp and another containing all residual parts, herein referred to as "DelRest". Additionally, we planned to integrate D. acidovorans's methyl-malonyl-CoA-pathway, which is a crucial co-factor for one of the PKS domains and hence, for successful delftibactin production. Because we were unfortunately not able to clone DelH and thus the entire pathway after several attempts, we decided to create a third plasmid not only containing the pathway, but as well a permeability device for enabeling our bacteria to secrete the desired product. These three plasmids were constructed in different subgroups and therefore have separated lab journals. Follow the links below to discover more!
Del H
The largest part of the pathway was amplified in subdivided fragments and subsequently assembled to a 23 kbp plasmid, which was later co-transformed with the other plasmids.
Del Rest
"DelRest" consists of the genes DelA, DelB, DelC, DelD, DelE, DelF, DelG, DelL, DelO and DelP, 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.
Delftibactin
We successfully expressed and purified Delftibactin in its natural host D. acidovorans. By dissolving gold from electronic waste and its recovery by D. acidovorans, we have successfully established a method enabling biological cost- and energy-efficient recycling of pure gold from electronic waste using delftibactin.
