Part:BBa_K1951011

The purpose of this biobrick is to produce desferrioxamine B siderophore by a controled manner, for controlled potential toxicity.

This biobrick was made from 6 parts:

Available in the registry :

• BBa_I0500
• BBa_B0034

Designed by our team :

• BBa_K1951000
• BBa_K1951001
• BBa_K1951002
• BBa_K1951003

Modele of the siderophore cycle in E. coli

Siderophores are small, high-affinity iron chelating compounds secreted by microorganisms such as bacteria, fungi and grasses. Siderophores are amongst the strongest soluble Fe3+ binding agents known.

A precious metal binder

Previous work showed that siderophores are able to catch other metals by default. Especifically, many articles showed a high affinity of Desferrioxamine B ( produce by Streptomyces coelicolor) for tetravalent metal ions like platinum. These results encouraged us to make a biobrick coding the sequence corresponding to the 4 enzymes involved on the metabolic pathway of Desferrioxamine B. E. coli was used to produce this biobrick. Produce a gram positive bacteria pathway in a gram negative bacteria is restrictive , considering the risk of toxicity for the conductress bacteria. To counter this potential issue, we regulate trasnscription using the control of an inductible promotor (pBAD/araC).

A medical treatment

Desferrioxamine B is a really well referenced siderophore.

Deferoxamine acts by binding free iron in the bloodstream and enhancing its elimination in the urine. By removing excess iron, the agent reduces the damage done to various organs and tissues, such as the liver. Also, it speeds healing of nerve damage (and minimizes the extent of recent nerve trauma).

• It is currently used of on the World Health Organization's List of Essential Medicines, the most important medications needed in a basic health system. But it is used in the treatment of many others diseases as well.

• Desferrioxamine B may modulate expression and release of inflammatory mediators by specific cell types.

Design sum up

To ensure the good protein quality and solubility :

• Coding sequence we have designed previouslyBBa_K1951000BBa_K1951001BBa_K1951002BBa_K1951003 have been optimized for E. coli
• The promotor and RBS we have used I0500 and B0034 were designed and tested in previous studies and demonstrate as inductible. Inducted, protomer allows a medium level expression in E.coli

SLIC oligo table

DesA slic forward	cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGCGCTCGCACTTGCTT
desA slic reverse	ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAGGAGGCACGGTC
desB slic forward	cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGGGTATTGGTCTTGGG
desB slic reverse	ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTACACTGCAAATTC
desC slic forward	cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGTCTCGCCTTTCCACG
desC slic reverse	ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAGGCTGAAACCGC
desD slic forward	cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGAGTTTAGCTGATGCA
des D slic reverse	ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAACGCCCGGCTAA

Experience sum up

To test the correct functioning of this biobrick we have performed three different types of experiment.

• Proteins production checked by SDS PAGE
• Mesure of cadaverine production tested by HPLC

Proteins production

Results showed the production of the 4 proteins DesA, DesB, DesC and DesD, all involved in the desferrioxamine B biosynthesis pathway as illustrated here.

HPLC test

We made a big composite part able to produce every proteins involved in the biosynthetic pathway of the desferrioxamine B